Multivalent binding composition for nucleic acid analysis

ABSTRACT

Multivalent binding compositions including a particle-nucleotide conjugate having a plurality of copies of a nucleotide attached to the particle are described. The multivalent binding compositions allow one to localize detectable signals to active regions of biochemical interaction, e.g., sites of protein-protein interaction, protein-nucleic acid interaction, nucleic acid hybridization, or enzymatic reaction, and can be used to identify sites of base incorporation in elongating nucleic acid chains during polymerase reactions and to provide improved base discrimination for sequencing and array based applications.

CROSS REFERENCE

This application is a continuation of U.S. patent application Ser. No.16/936,121 filed Jul. 22, 2020, which is a continuation of U.S. patentapplication Ser. No. 16/579,794 filed Sep. 23, 2019, now U.S. Pat. No.10,768,173 issued Sep. 8, 2020, which claims the benefit of U.S.Provisional Application No. 62/897,172 filed Sep. 6, 2019, each of whichis incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to multivalent bindingcompositions and their use in analyzing nucleic acid molecules. Inparticular, the invention relates to a multivalent binding compositionhaving multiple copies of a nucleotide attached to a particle whicheffectively increases the local concentration of the nucleotide andenhances the binding signals. The multivalent binding composition can beapplied, for example, in the field of sequencing and biosensormicroarrays.

BACKGROUND

Nucleic acid sequencing can be used to obtain information in a widevariety of biomedical contexts, including diagnostics, prognostics,biotechnology, and forensic biology. Various sequencing methods havebeen developed including Maxam-Gilbert sequencing and chain-terminationmethods, or de novo sequencing methods including shotgun sequencing andbridge PCR, or next-generation methods including polony sequencing, 454pyrosequencing, Illumina sequencing, SOLiD sequencing, Ion Torrentsemiconductor sequencing, HeliScope single molecule sequencing, SMRT®sequencing, and others. Despite advances in DNA sequencing, manychallenges still remain unaddressed. The present disclosure providesnovel solutions and approaches to addressing many of the shortcomings ofexisting technologies.

SUMMARY

In some embodiments the present disclosure provides method ofdetermining the identity of a nucleotide in a target nucleic acidcomprising the steps, without regard to any particular order ofoperations, 1) providing a composition comprising: a target nucleic acidcomprising two or more repeats of an identical sequence; two or moreprimer nucleic acids complementary to one or more regions of said targetnucleic acid; and two or more polymerase molecules; 2) contacting saidcomposition with a multivalent binding composition comprising apolymer-nucleotide conjugate under conditions sufficient to allow abinding complex to be formed between said polymer-nucleotide conjugateand the composition of step (a), wherein the polymer-nucleotideconjugate comprises two or more copies of a nucleotide and optionallyone or more detectable labels; and 3) detecting said binding complex,thereby establishing the identity of said nucleotide in the targetnucleic acid polymer. In some further embodiments, the presentdisclosure provides said method, wherein the target nucleic acid is DNA,and/or wherein the target nucleic acid has been replicated, such as byany commonly practiced method of DNA replication or amplification, suchas rolling circle amplification, bridge amplification, helicasedependent amplification, isothermal bridge amplification, rolling circlemultiple displacement amplification (RCA/MDA) and/or recombinase basedmethods of replication or amplification. In some further embodiments,the present disclosure provides said method, wherein the detectablelabel is a fluorescent label and/or wherein detecting the complexcomprises a fluorescence measurement. In some further embodiments, thepresent disclosure provides said method wherein the multivalent bindingcomposition comprises one type of polymer-nucleotide conjugate, whereinthe multivalent binding composition comprises two or more types ofpolymer-nucleotide conjugates, and/or wherein each type of the two ormore types of polymer-nucleotide conjugates comprises a different typeof nucleotide. In some embodiments, the present disclosure provides saidmethod wherein the binding complex further comprises a blockednucleotide, especially wherein the blocked nucleotide is a3′-O-azidomethyl, a 3′-O-alkyl hydroxylamino or 3′-O-methyl nucleotide.In some further embodiments, the present disclosure provides said methodwherein the contacting is done in the presence of strontium ions,magnesium ions, and/or calcium ions. In some embodiments, the presentdisclosure provides said method wherein the polymerase molecule iscatalytically inactive, such as where the polymerase molecule beenrendered catalytically inactive by mutation, by chemical modification,or by the absence of a necessary ion or cofactor. In some embodiments,the present disclosure also provides said method wherein the polymerasemolecule is catalytically active, and/or wherein the binding complexdoes not comprise a blocked nucleotide. In some embodiments, the presentdisclosure provides said method wherein the binding complex has apersistence time of greater than 2 seconds and/or wherein the method isor may be carried out at a temperature of at or above 15° C., at orabove 20° C., at or above 25° C., at or above 35° C., at or above 37°C., at or above 42° C. at or above 55° C. at or above 60° C., or at orabove 72° C., or within a range defined by any of the foregoing. In someembodiments, the present disclosure provides said method wherein thebinding complex is deposited on, attached to, or hybridized to, asurface showing a contrast to noise ratio in the detecting step ofgreater than 20. In some embodiments, the present disclosure providessaid method wherein the composition is deposited under buffer conditionsincorporating a polar aprotic solvent. In some embodiments, the presentdisclosure provides said method wherein the contacting is performedunder a condition that stabilizes said binding complex when saidnucleotide is complementary to a next base of said target nucleic acid,and destabilizes said binding complex when said nucleotide is notcomplementary to said next base of said target nucleic acid. In someembodiments, the present disclosure provides said method wherein saidpolymer-nucleotide conjugate comprises a polymer having a plurality ofbranches and said plurality of copies of said first nucleotide areattached to said branches, especially wherein said first polymer has astar, comb, cross-linked, bottle brush, or dendrimer configuration. Insome embodiments, the present disclosure provides said method whereinsaid polymer-nucleotide conjugate comprises one or more binding groupsselected from the group consisting of avidin, biotin, affinity tag, andcombinations thereof. In some embodiments, the present disclosureprovides said method further comprising a dissociation step thatdestabilizes said binding complex formed between the composition of (a)and the polymer-nucleotide conjugate to remove said polymer-nucleotideconjugate. In some embodiments, the present disclosure provides saidmethod further comprising an extension step to incorporate into saidprimer nucleic acid a nucleotide that is complementary to said next baseof the target nucleic acid, and optionally wherein the extension stepoccurs currently as or after said dissociation step.

In some embodiments, the present disclosure provides a compositioncomprising a branched polymer having two or more branches and two ormore copies of a nucleotide, wherein said nucleotide is attached to afirst plurality of said branches or arms, and optionally, wherein one ormore interaction moieties are attached to a second plurality of saidbranches or arms. In some embodiments, said composition may furthercomprise one or more labels on the polymer. In some embodiments, thepresent disclosure provides said composition wherein the nucleoside hasa surface density of at least 4 nucleotides per polymer. In someembodiments, the present disclosure provides said composition comprisingor incorporating a nucleotide or nucleotide analog that is modified soas to prevent its incorporation into an extending nucleic acid chainduring a polymerase reaction. In some embodiments, said composition maycomprise or incorporate a nucleotide or nucleotide analog that isreversibly modified so as to prevent its incorporation into an extendingnucleic acid chain during a polymerase reaction. In some embodiments,the present disclosure provides said composition wherein one or morelabels comprise a fluorescent label, a FRET donor, and/or a FRETacceptor. In some embodiments, said composition may comprise 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more branches or arms, or 2,4, 8, 16, 32, 64, or more, branches or arms. In some embodiments, thebranches or arms may radiate from a central moiety. In some embodiments,said composition may comprise one or more interaction moieties, whichinteraction moieties may comprise avidin or streptavidin; a biotinmoiety; an affinity tag; an enzyme, antibody, minibody, receptor, orother protein; a non-protein tag; a metal affinity tag, or anycombination thereof. In some embodiments, the present disclosureprovides said composition wherein the polymer comprises polyethyleneglycol, polypropylene glycol, polyvinyl acetate, polylactic acid, orpolyglycolic acid. In some embodiments, the present disclosure providessaid composition wherein the nucleotide or nucleotide analog is attachedto the branch or arm through a linker; and especially wherein the linkercomprises PEG, and wherein the PEG moiety has an average molecularweight of about 1K, about 2K, about 3K, about 4K, about 5K, about 10K,about 15K, about 20K, about 50K, about 100K, about 150K, or about 200K,or greater than about 200K. In some embodiments, the present disclosureprovides said composition wherein the linker comprises PEG, and whereinthe PEG moiety has an average molecular weight of between about 5K andabout 20K. In some embodiments, the present disclosure provides saidcomposition wherein at least one nucleotide or nucleotide analogcomprises a deoxyribonucleotide, a ribonucleotide, adeoxyribonucleoside, or a ribonucleoside; and/or wherein the nucleotideor nucleotide analog is conjugated to the linker through the 5′ end ofthe nucleotide or nucleotide analog. In some embodiments, the presentdisclosure provides said composition wherein one of the nucleotides ornucleotide analogs comprises deoxyadenosine, deoxyguanosine, thymidine,deoxyuridine, deoxycytidine, adenosine, guanosine, 5-methyl-uridine,and/or cytidine; and wherein the length of the linker is between 1 and1,000 nm. In some embodiments, the present disclosure provides saidcomposition wherein at least one nucleotide or nucleotide analog is anucleotide that has been modified to inhibit elongation during apolymerase reaction or a sequencing reaction, such as wherein the atleast one nucleotide or nucleotide analog is a nucleotide that lacks a3′ hydroxyl group; a nucleotide that has been modified to contain ablocking group at the 3′ position; and/or a nucleotide that has beenmodified with a 3′-O-azido group, a 3′-O-azidomethyl group, a 3′-O-alkylhydroxylamino group, a 3′-phosphorothioate group, a 3′-O-malonyl group,or a 3′-O-benzyl group. In some embodiments, the present disclosureprovides said composition wherein at least one nucleotide or nucleotideanalog is a nucleotide that has not been modified at the 3′ position.

In some embodiments, the present disclosure provides a method ofdetermining the sequence of a nucleic acid molecule comprising thesteps, without regard to any particular order, of 1) providing a nucleicacid molecule comprising a template strand and a complementary strandthat is at least partially complementary to the template strand; 2)contacting the nucleic acid molecule with the one or more nucleic acidbinding composition according to any one of claims 31-42; 3) detectingbinding of the nucleic acid binding composition to the nucleic acidmolecule, and 4) determining an identity of a terminal nucleotide to beincorporated into said complementary strand of said nucleic acidmolecule. In some embodiments, the present disclosure provides saidmethod, further comprising incorporating said terminal nucleotide intosaid complementary strand, and repeating said contacting, detecting, andincorporating steps for one or more additional iterations, therebydetermining the sequence of said template strand of said nucleic acidmolecule. In some embodiments, the present disclosure provides saidmethod, wherein said nucleic acid molecule is tethered to a solidsupport; and especially wherein the solid support comprises a glass orpolymer substrate, at least one hydrophilic polymer coating layer, and aplurality of oligonucleotide molecules attached to at least onehydrophilic polymer coating layer. In some embodiments, the presentdisclosure provides said method, further comprising embodiments whereinat least one hydrophilic polymer coating layer comprises PEG; and/orwherein at least one hydrophilic polymer layer comprises a branchedhydrophilic polymer having at least 8 branches. In some embodiments, thepresent disclosure provides said method, wherein the plurality ofoligonucleotide molecules is present at a surface density of at least500 molecules/mm², at least 1,000 molecules/mm², at least 5,000molecules/mm², at least 10,000 molecules/mm², at least 20,000molecules/mm², at least 50,000 molecules/mm², at least 100,000molecules/mm², or at least 500,000 molecules/mm². In some embodiments,the present disclosure provides said method, wherein said nucleic acidmolecule has been clonally-amplified on a solid support. In someembodiments, the present disclosure provides said method, wherein theclonal amplification comprises the use of a polymerase chain reaction(PCR), multiple displacement amplification (MDA), transcription-mediatedamplification (TMA), nucleic acid sequence-based amplification (NASBA),strand displacement amplification (SDA), real-time SDA, bridgeamplification, isothermal bridge amplification, rolling circleamplification, circle-to-circle amplification, helicase-dependentamplification, recombinase-dependent amplification, single-strandedbinding (SSB) protein-dependent amplification, or any combinationthereof. In some embodiments, the present disclosure provides saidmethod, wherein the one or more nucleic acid binding compositions arelabeled with fluorophores and the detecting step comprises use offluorescence imaging; and especially wherein the fluorescence imagingcomprises dual wavelength excitation/four wavelength emissionfluorescence imaging. In some embodiments, the present disclosureprovides said method, wherein four different nucleic acid bindingcompositions, each comprising a different nucleotide or nucleotideanalog, are used to determine the identity of the terminal nucleotide,wherein the four different nucleic acid binding compositions are labeledwith separate respective fluorophores, and wherein the detecting stepcomprises simultaneous excitation at a wavelength sufficient to exciteall four fluorophores and imaging of fluorescence emission atwavelengths sufficient to detect each respective fluorophore. In someembodiments, the present disclosure provides said method, wherein fourdifferent nucleic acid binding compositions, each comprising a differentnucleotide or nucleotide analog, are used to determine the identity ofthe terminal nucleotide, wherein the four different nucleic acid bindingcompositions are labeled with Cy3, Cy3.5, Cy5, and Cy5.5 respectively,and wherein the detecting step comprises simultaneous excitation at anytwo of 532 nm, 568 nm and 633 nm, and imaging of fluorescence emissionat about 570 nm, 592 nm, 670 nm, and 702 nm respectively; and/or whereinthe fluorescence imaging comprises dual wavelength excitation/dualwavelength emission fluorescence imaging. In some embodiments, thepresent disclosure provides said method, wherein four different nucleicacid binding compositions, each comprising a different nucleotide ornucleotide analog, are used to determine the identity of the terminalnucleotide, wherein one, two, three, or four different nucleic acidbinding compositions are respectively labeled, each with a with distinctfluorophore or set of fluorophores, and wherein the detecting stepcomprises simultaneous excitation at a wavelength sufficient to exciteone, two, three, or four fluorophores or sets of fluorophores, andimaging of fluorescence emission at wavelengths sufficient to detecteach respective fluorophore. In some embodiments, the present disclosureprovides said method, wherein three different nucleic acid bindingcompositions, each comprising a different nucleotide or nucleotideanalog, are used to determine the identity of the terminal nucleotide,wherein one, two, or three different nucleic acid binding compositionsare respectively labeled, each with a with distinct fluorophore or setof fluorophores, and wherein the detecting step comprises simultaneousexcitation at a wavelength sufficient to excite one, two, or three,fluorophores or sets of fluorophores, and imaging of fluorescenceemission at wavelengths sufficient to detect each respectivefluorophore, and wherein detection of the fourth nucleotide isdetermined or determinable with reference to the location of “dark” orunlabeled spots or target nucleotides. In some embodiments, the presentdisclosure provides said method, wherein the multivalent bindingcomposition consists of three types of polymer-nucleotide conjugates andwherein each type of the three types of polymer-nucleotide conjugatescomprises a different type of nucleotide. In some embodiments, thepresent disclosure provides said method, wherein the detection of thebinding complex is performed in the absence of unbound or solution-bornepolymer nucleotide conjugates.

In some embodiments, the present disclosure provides said method,wherein four different nucleic acid binding compositions, or threedifferent nucleic acid binding compositions, each comprising a differentnucleotide or nucleotide analog, are used to determine the identity ofthe terminal nucleotide, wherein one of the four or three differentnucleic acid binding compositions is labeled with a first fluorophore,one is labeled with a second fluorophore, one is labeled with both thefirst and second fluorophore, and one is not labeled or is absent, andwherein the detecting step comprises simultaneous excitation at a firstexcitation wavelength and a second excitation wavelength and images areacquired at a first fluorescence emission wavelength and a secondfluorescence emission wavelength. In some embodiments, the presentdisclosure provides said method, wherein the first fluorophore is Cy3,the second fluorophore is Cy5, the first excitation wavelength is 532 nmor 568 nm, the second excitation wavelength is 633 nm, the firstfluorescence emission wavelength is about 570 nm, and the secondfluorescence emission wavelength is about 670 nm. In some embodiments,the present disclosure provides said method, wherein the detection labelcan comprise one or more portions of a FRET pair, such that multipleclassifications can be performed under a single excitation and imagingstep. In some embodiments, the present disclosure provides said method,wherein a sequencing reaction cycle comprising the contacting,detecting, and incorporating/extending steps is performed in less than30 minutes in less than 20 minutes, or in less than 10 minutes. In someembodiments, the present disclosure provides said method, wherein anaverage Q-score for base calling accuracy over a sequencing run isgreater than or equal to 30, and/or greater than or equal to 40. In someembodiments, the present disclosure provides said method, wherein atleast 50%, at least 60%, at least 70%, at least 80%, or at least 90% ofthe terminal nucleotides identified have a Q-score of greater than 30and/or greater than or equal to 40. In some embodiments, the presentdisclosure provides said method, herein at least 95% of the terminalnucleotides identified have a Q-score of greater than 30.

In some embodiments, the present disclosure provides a reagentcomprising one or more nucleic acid binding compositions as disclosedherein and a buffer. For example, in some embodiments, the presentdisclosure provides a reagent, wherein said reagent comprises 1, 2, 3,4, or more nucleic acid binding compositions, wherein each nucleic acidbinding composition comprises a single type of nucleotide. In someembodiments, a reagent of the present disclosure comprises 1, 2, 3, 4,or more nucleic acid binding compositions, wherein each nucleic acidbinding composition comprises a single type of nucleotide or nucleotideanalog, and wherein said nucleotide or nucleotide analog mayrespectively correspond to one or more from the group consisting of ATP,ADP, AMP, dATP, dADP, and dAMP; one or more from the group consisting ofTTP, TDP, TMP, dTTP, dTDP, dTMP, UTP, UDP, UMP, dUTP, dUDP, and dUMP;one or more from the group consisting of CTP, CDP, CMP, dCTP, dCDP, anddCMP; and one or more from the group consisting of GTP, GDP, GMP, dGTP,dGDP, and dGMP. In some other examples or some further examples, thepresent disclosure provides a reagent comprising or further comprising1, 2, 3, 4, or more nucleic acid binding compositions, wherein eachnucleic acid binding composition comprises a single type of nucleotideor nucleotide analog, and wherein said nucleotide or nucleotide analogmay respectively correspond to one or more from the group consisting ofATP, ADP, AMP, dATP, dADP, dAMP, TTP, TDP, TMP, dTTP, dTDP, dTMP, UTP,UDP, UMP, dUTP, dUDP, dUMP, CTP, CDP, CMP, dCTP, dCDP, dCMP, GTP, GDP,GMP, dGTP, dGDP, and dGMP.

In some embodiments, the present disclosure provides a kit comprisingthe nucleic acid binding composition as disclosed herein and/or areagent as disclosed herein, and/or one or more buffers; andinstructions for the use thereof.

In some embodiments, the present disclosure provides a system forperforming the method or methods disclosed herein, comprising a nucleicacid binding composition as disclosed herein, and/or a reagent a, sdisclosed herein. In some embodiments, a system is configured toiteratively perform the sequential contacting of said tethered nucleicacid molecules with said nucleic acid binding composition and/or saidreagent; and for the detection of binding of the nucleic acid bindingcompositions to the one or more nucleic acid molecules.

In some embodiments, the present disclosure provides a compositioncomprising a particle, said particle comprising a plurality of enzyme orprotein binding substrates, wherein the enzyme or protein bindingsubstrates bind with one or more enzymes or proteins to form one or morebinding complexes, and wherein said binding may be monitored oridentified by observation of the location, presence, or persistence ofone or more binding complexes. In some embodiments, said particle maycomprise a polymer, branched polymer, dendrimer, liposome, micelle,nanoparticle, or quantum dot. In some embodiments, said substrate maycomprise a nucleotide, a nucleoside, a nucleotide analog, or anucleoside analog. In some embodiments, the enzyme or protein bindingsubstrate may comprise an agent that can bind with a polymerase. In someembodiments, the enzyme or protein may comprise a polymerase. In someembodiments, said observation of the location, presence, or persistenceof one or more binding complexes may comprise fluorescence detection. Insome embodiments, the present disclosure provides a compositioncomprising multiple distinct particles as disclosed herein, wherein eachparticle comprises a single type of nucleoside or nucleoside analog, andwherein each nucleoside or nucleoside analog is associated with afluorescent label of a detectably different emission or excitationwavelength. In some embodiments, the present disclosure provides saidcomposition further comprising one or more labels on the particle. Insome embodiments, the present disclosure provides said compositionwherein the nucleoside or nucleoside analog has a surface density of atleast 4 nucleosides or nucleoside analogs. In some embodiments, thepresent disclosure provides said composition wherein the nucleoside ornucleoside analog has a surface density of between 0.001 and 1,000,000per μm², between 0.01 and 1,000,000 per μm², between 0.1 and 1,000,000per μm², between 1 and 1,000,000 per μm², between 10 and 1,000,000 perμm², between 100 and 1,000,000 per μm², between 1,000 and 1,000,000 perμm², between 1,000 and 100,000 per μm², between 10,000 and 100,000 perμm², or between 50,000 and 100,000 per μm², or within a range defined bynay two of the foregoing values In some embodiments, the presentdisclosure provides said composition wherein the nucleoside ornucleoside analog is present within a nucleotide or nucleotide analog.In some embodiments, the present disclosure provides said compositionwherein the composition comprises or incorporates a nucleotide ornucleotide analog that is modified so as to prevent its incorporationinto an extending nucleic acid chain during a polymerase reaction. Insome embodiments, the present disclosure provides said compositionwherein the composition comprises or incorporates a nucleotide ornucleotide analog that is reversibly modified so as to prevent itsincorporation into an extending nucleic acid chain during a polymerasereaction. In some embodiments, the present disclosure provides saidcomposition wherein one or more labels comprise a fluorescent label, aFRET donor, and/or a FRET acceptor. In some embodiments, the presentdisclosure provides said composition wherein the substrate is attachedto the particle through a linker. In some embodiments, the presentdisclosure provides said composition wherein at least one nucleotide ornucleotide analog is a nucleotide that has been modified to inhibitelongation during a polymerase reaction or a sequencing reaction, suchas, for example, a nucleotide that lacks a 3′ hydroxyl group; anucleotide that has been modified to contain a blocking group at the 3′position; a nucleotide that has been modified with a 3′-O-azido group, a3′-0-azidomethyl group, a 3′-O-alkyl hydroxylamino group, a3′-phosphorothioate group, a 3′-O-malonyl group, or a 3′-O-benzyl group;and/or a nucleotide that has not been modified at the 3′ position.

In some embodiments, the present disclosure provides a method ofdetermining the sequence of a nucleic acid molecule comprising thesteps, without regard to order, of 1) providing a nucleic acid moleculecomprising a template strand and a complementary strand that is at leastpartially complementary to the template strand; 2) contacting thenucleic acid molecule with the one or more nucleic acid bindingcomposition according to any one of claims 31-70 and 100-121; 3)detecting binding of the nucleic acid binding composition to the nucleicacid molecule, and 4) determining an identity of a terminal nucleotideto be incorporated into said complementary strand of said nucleic acidmolecule. In some embodiments, said method may further compriseincorporating said terminal nucleotide into said complementary strand,and repeating said contacting, detecting, and incorporating steps forone or more additional iterations, thereby determining the sequence ofsaid template strand of said nucleic acid molecule. In some embodiments,the present disclosure provides said method wherein said nucleic acidmolecule has been clonally-amplified on a solid support. In someembodiments, the present disclosure provides said method wherein theclonal amplification comprises the use of a polymerase chain reaction(PCR), multiple displacement amplification (MDA), transcription-mediatedamplification (TMA), nucleic acid sequence-based amplification (NASBA),strand displacement amplification (SDA), real-time SDA, bridgeamplification, isothermal bridge amplification, rolling circleamplification, circle-to-circle amplification, helicase-dependentamplification, recombinase-dependent amplification, single-strandedbinding (SSB) protein-dependent amplification, or any combinationthereof. In some embodiments, the present disclosure provides saidmethod wherein a sequencing reaction cycle comprising the contacting,detecting, and incorporating steps is performed in less than 30 minutes,less than 20 minutes, or in less than 10 minutes. In some embodiments,the present disclosure provides said method wherein an average Q-scorefor base calling accuracy over a sequencing run is greater than or equalto 30, or greater than or equal to 40. In some embodiments, the presentdisclosure provides said method wherein at least 50%, at least 60%, atleast 70%, at least 80%, or at least 90% of the terminal nucleotidesidentified have a Q-score of greater than 30; or greater than 40. Insome embodiments, the present disclosure provides said method wherein atleast 95% of the terminal nucleotides identified have a Q-score ofgreater than 30.

In some embodiments, the present disclosure provides a reagentcomprising one or more nucleic acid binding compositions as disclosedherein, and a buffer. In some embodiments, the present disclosureprovides said reagent, wherein said reagent comprises 1, 2, 3, 4, ormore nucleic acid binding compositions, wherein each nucleic acidbinding composition comprises a single type of nucleotide or nucleotideanalog, and wherein said nucleotide or nucleotide analog comprises anucleotide, nucleotide analog, nucleoside, or nucleoside analog. In someembodiments, the present disclosure provides said method wherein saidreagent comprises 1, 2, 3, 4, or more nucleic acid binding compositions,wherein each nucleic acid binding composition comprises a single type ofnucleotide or nucleotide analog, and wherein said nucleotide ornucleotide analog may respectively correspond to one or more from thegroup consisting of ATP, ADP, AMP, dATP, dADP, and dAMP; one or morefrom the group consisting of TTP, TDP, TMP, dTTP, dTDP, dTMP, UTP, UDP,UMP, dUTP, dUDP, and dUMP; one or more from the group consisting of CTP,CDP, CMP, dCTP, dCDP, and dCMP; and one or more from the groupconsisting of GTP, GDP, GMP, dGTP, dGDP, and dGMP. In some embodiments,the present disclosure provides said method wherein said reagentcomprises 1, 2, 3, 4, or more nucleic acid binding compositions, whereineach nucleic acid binding composition comprises a single type ofnucleotide or nucleotide analog, and wherein said nucleotide ornucleotide analog may respectively correspond to one or more from thegroup consisting of ATP, ADP, AMP, dATP, dADP, dAMP TTP, TDP, TMP, dTTP,dTDP, dTMP, UTP, UDP, UMP, dUTP, dUDP, dUMP, CTP, CDP, CMP, dCTP, dCDP,dCMP, GTP, GDP, GMP, dGTP, dGDP, and dGMP.

In some embodiments, the present disclosure provides a kit comprisingany of the compositions disclosed herein; and/or any of the reagentsdisclosed herein; one or more buffers; and instructions for the usethereof.

In some embodiments, the present disclosure provides a system forperforming any of the methods disclosed herein; wherein said methods maycomprise use of any of the compositions as disclosed herein; and/or anyof the reagents as disclosed herein; one or more buffers, and one ormore nucleic acid molecules optionally tethered or attached to a solidsupport, wherein said system is configured to iteratively perform forthe sequential contacting of said nucleic acid molecules with saidcomposition and/or said reagent; and for the detection of binding of thenucleic acid binding compositions to the one or more nucleic acidmolecules.

In some embodiments, the present disclosure provides a composition asdisclosed herein for use in increasing the contrast to noise ratio (CNR)of a labeled nucleic acid complex bound to or associated with a surface.

In some embodiments, the present disclosure provides a composition asdisclosed herein for use in establishing or maintaining control over thepersistence time of a signal from a labeled nucleic acid complex boundto or associated with a surface.

In some embodiments, the present disclosure provides a composition asdisclosed herein for use in establishing or maintaining control over thepersistence time of a fluorescence, luminescence, electrical,electrochemical, colorimetric, radioactive, magnetic, or electromagneticsignal from a labeled nucleic acid complex bound to or associated with asurface.

In some embodiments, the present disclosure provides a composition asdisclosed herein for use in increasing the specificity, accuracy, orread length of a nucleic acid sequencing and/or genotyping application.

In some embodiments, the present disclosure provides a composition asdisclosed herein for use in increasing the specificity, accuracy, orread length in a sequencing by binding, sequencing by synthesis, singlemolecule sequencing, or ensemble sequencing method.

In some embodiments, the present disclosure provides a reagent asdisclosed herein for use in increasing the contrast to noise ratio (CNR)of a labeled nucleic acid complex bound to or associated with a surface.

In some embodiments, the present disclosure provides a reagent asdisclosed herein for use in establishing or maintaining control over thepersistence time of a signal from a labeled nucleic acid complex boundto or associated with a surface.

In some embodiments, the present disclosure provides a reagent asdisclosed herein for use in establishing or maintaining control over thepersistence time of a fluorescence, luminescence, electrical,electrochemical, colorimetric, radioactive, magnetic, or electromagneticsignal from a labeled nucleic acid complex bound to or associated with asurface.

In some embodiments, the present disclosure provides a reagent asdisclosed herein for use in increasing the specificity, accuracy, orread length of a nucleic acid sequencing and/or genotyping application.

In some embodiments, the present disclosure provides a reagent asdisclosed herein for use in increasing the specificity, accuracy, orread length in a sequencing by binding, sequencing by synthesis, singlemolecule sequencing, or ensemble sequencing method.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference in their entirety tothe same extent as if each individual publication, patent, or patentapplication was specifically and individually indicated to beincorporated by reference in its entirety. In the event of a conflictbetween a term herein and a term in an incorporated reference, the termherein controls.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIGS. 1A-1H illustrate the steps utilizing a non-limiting examples ofmultivalent binding composition for sequencing a target nucleic acid:FIG. 1A illustrates a non-limiting example 4-of attaching target nucleicacid to a surface; FIG. 1B illustrates clonally the target nucleic acidto form clusters of amplified target nucleic acid molecules; FIG. 1Cillustrates a non-limiting example of priming the target nucleic acid toproduce a primed target nucleic acid; FIG. 1D illustrates a non-limitingexample of contacting the primed target nucleic acid to the multivalentbinding composition and polymerase to form a binding complex; FIG. 1Eillustrates a non-limiting example of the images of the binding complexcaptured on the surface; FIG. 1F illustrates a non-limiting example ofextending the primer strand by one nucleotide; FIG. 1G illustrates anon-limiting example of another cycle of contacting the primed targetnucleic acid to the multivalent binding composition and polymerase toform a binding complex; and FIG. 1H illustrates non-limiting examples ofthe images of binding complex captured on the surface in subsequentsequencing cycles.

FIG. 2A-2B illustrate a non-limiting example of detecting target nucleicacid using the polymer-nucleotide conjugates. FIG. 2A shows the step ofcontacting the polymerase and polymer-nucleotide conjugates to somenucleic acid molecules; FIG. 2B shows the binding complex formed betweenthe polymerase, polymer-nucleotide conjugates, and the target nucleicacid molecules.

FIG. 3 shows a flow chart outlining the steps for sequencing a targetnucleic acid and extending the primer strand through a single baseaddition.

FIG. 4 shows a flow chart outlining the steps for sequencing a targetnucleic acid and extending the primer strand through incorporating thenucleotide on the particle-nucleotide conjugate.

FIG. 5A-5C shows schematic representations of non-limiting examples ofvarying configurations of the polymer-nucleotide conjugates: FIG. 5Ashows polymer-nucleotide conjugates having various multi-armconfigurations; FIG. 5B shows a polymer-nucleotide conjugate having thepolymer branch radiating from the center; and FIG. 5C showspolymer-nucleotide conjugates having the binding moiety biotin.

FIG. 6 shows a generalized graphical depiction of the increase in signalintensity that has been observed during binding, persistence, andwashing and removal of multivalent substrates.

FIGS. 7A-7G show fluorescence images of multivalent polyethylene glycol(PEG) polymer-nucleotide (base-labeled) conjugates, having an effectivenucleotide concentration of 500 nM and varying PEG branch length, aftercontacting to a support surface comprising DNA templates (comprising Gor A as the first base; prepared using rolling circle amplification(RCA)) in an exposure buffer comprising 20 nM Klenow polymerase and 2.5mM Sr⁺². Images were acquired after washing with an imaging bufferhaving the same composition as the exposure buffer but lackingnucleotides and polymeras. Panels show images obtained using multivalentPEG-nucleotide ligands with arm lengths as follows. FIG. 7A: 1K PEG.FIG. 7B: 2K PEG. FIG. 7C: 3K PEG. FIG. 7D: 5K PEG. FIG. 7E: 10K PEG.FIG. 7F: 20K PEG. FIG. 7G shows images obtained using 10K PEG and aninactive klenow polymerase comprising the mutation D882H. FIG. 7H showsimages obtained using 10K PEG and an inactive klenow polymerasecomprising the mutation D882E. FIG. 7I shows images obtained using 10KPEG and an inactive klenow polymerase comprising the mutation D882A.FIG. 7G shows images obtained using 10K PEG and an active wild typeklenow polymerase.

FIG. 8 shows a quantitative representation of the fluorescenceintensities in the images shown in FIGS. 7A-7F, separated by colorvalue, with orange trace corresponding to the red label (Cy3 label; Abases) and blue trace corresponding to the green label (Cy5 label; Gbases).

FIGS. 9A-9J show fluorescence images of the steps in a sequencingreaction using multivalent PEG-substrate compositions. FIG. 9A. Red andgreen fluorescent images post exposure of DNA RCA templates (G and Afirst base) to 500 nM base labeled nucleotides (A-Cy3 and G-Cy5) inexposure buffer containing 20 nM Klenow polymerase and 2.5 mM Sr⁺².Images were collected after washing with imaging buffer with the samecomposition as the exposure buffer but containing no nucleotides orpolymerase. Contrast was scaled to maximize visualization of the dimmestsignals, but no signals persisted following washing with imaging buffer(a. inset). FIGS. 9B-9E: fluorescence images showing multivalentPEG-nucleotide (base-labeled) ligands PB1 (FIG. 9B), PB2 (FIG. 9C), PB3(FIG. 9D), and PB5 (FIG. 9E) having an effective nucleotideconcentration of 500 nM after mixing in the exposure buffer and imagingin the imaging buffer as described above. FIG. 9F: fluorescence imageshowing multivalent PEG-nucleotide (base-labeled) ligand PB5 at 2.5 uMafter mixing in the exposure buffer and imaging in the imaging buffer asabove. FIGS. 9G-9I. Fluorescence images showing further basediscrimination by exposure of the multivalent binding composition toinactive mutants of klenow polymerase (FIG. 9G. D882H; FIG. 9H. D882E;FIG. 9I. D882A) vs. the wild type Klenow (control) enzyme (FIG. 9J).

FIGS. 10A-10B show the efficacy of the multivalent reporter compositionsin determining the base sequence of a DNA sequence over 5 sequencingcycles: FIG. 10A shows images and expected sequences for templates takenafter each sequencing cycle; and FIG. 10B shows aligned sequencingresults utilizing the images taken in FIG. 10A.

FIG. 11 shows normalized fluorescence from multivalent substrates boundto DNA clusters as in FIG. 9, with the substrate complexes formed in thepresence (condition B) and absence (condition A) of Triton-X100(0.016%).

FIG. 12 shows normalized fluorescence of multivalent substrates and freenucleotides. (Top) Two replicates of a multivalent substrate bound toDNA clusters (Conditions A and B) vs. binding complexes formed usinglabeled free nucleotides (Condition C) after 1 minute; (Bottom)Timecourse of fluorescence from multivalent substrate complexes over thecourse of 60 min.

DETAILED DESCRIPTION I. Definitions

As used herein, “nucleic acid” (also referred to as a “polynucleotide”,“oligonucleotide”, ribonucleic acid (RNA), or deoxyribonucleic acid(DNA)) is a linear polymer of two or more nucleotides joined by covalentinternucleosidic linkages, or variants or functional fragments thereof.In naturally occurring examples of nucleic acids, the internucleosidelinkage is typically a phosphodiester bond. However, other examplesoptionally comprise other internucleoside linkages, such asphosphorothiolate linkages and may or may not comprise a phosphategroup. Nucleic acids include double- and single-stranded DNA, as well asdouble- and single-stranded RNA, DNA/RNA hybrids, peptide-nucleic acids(PNAs), hybrids between PNAs and DNA or RNA, and may also include othertypes of nucleic acid modifications.

As used herein, a “nucleotide” refers to a nucleotide, nucleoside, oranalog thereof. The nucleotide refers to both naturally occurring andchemically modified nucleotides and can include but are not limited to anucleoside, a ribonucleotide, a deoxyribonucleotide, a protein-nucleicacid residue, or derivatives. Examples of the nucleotide includes anadenine, a thymine, a uracil, a cytosine, a guanine, or residue thereof;a deoxyadenine, a deoxythymine, a deoxyuracil, a deoxycytosine, adeoxyguanine, or residue thereof; a adenine PNA, a thymine PNA, a uracilPNA, a cytosine PNA, a guanine PNA, or residue or equivalents thereof,an N- or C-glycoside of a purine or pyrimidine base (e.g., adeoxyribonucleoside containing 2-deoxy-D-ribose or ribonucleosidecontaining D-ribose).

“Complementary,” as used herein, refers to the topological compatibilityor matching together of interacting surfaces of a ligand molecule andits receptor. Thus, the receptor and its ligand can be described ascomplementary, and furthermore, the contact surface characteristics arecomplementary to each other.

“Branched polymer”, as used herein, refers to a polymer having aplurality of functional groups that help conjugate a biologically activemolecule such as a nucleotide, and the functional group can be either onthe side chain of the polymer or directly attaches to a central core orcentral backbone of the polymer. The branched polymer can have linearbackbone with one or more functional groups coming off the backbone forconjugation. The branched polymer can also be a polymer having one ormore sidechains, wherein the side chain has a site suitable forconjugation. Examples of the functional group include but are limited tohydroxyl, ester, amine, carbonate, acetal, aldehyde, aldehyde hydrate,alkenyl, acrylate, methacrylate, acrylamide, active sulfone, hydrazide,thiol, alkanoic acid, acid halide, isocyanate, isothiocyanate,maleimide, vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide,epoxide, glyoxal, dione, mesylate, tosylate, and tresylate.

“Polymerase,” as used herein, refers to an enzyme that contains anucleotide binding moiety and helps formation of a binding complexbetween a target nucleic acid and a complementary nucleotide. Thepolymerase can have one or more activities including, but not limitedto, base analog detection activities, DNA polymerization activity,reverse transcriptase activity, DNA binding, strand displacementactivity, and nucleotide binding and recognition. The polymerase caninclude catalytically inactive polymerase, catalytically activepolymerase, reverse transcriptase, and other enzymes containing anucleotide binding moiety.

“Persistence time,” as used herein, refers to the length of time that abinding complex, which is formed between the target nucleic acid, apolymerase, a conjugated or unconjugated nucleotide, remains stablewithout any binding component dissociates from the binding complex. Thepersistence time is indicative of the stability of the binding complexand strength of the binding interactions. Persistence time can bemeasured by observing the onset and/or duration of a binding complex,such as by observing a signal from a labeled component of the bindingcomplex. For example, a labeled nucleotide or a labeled reagentcomprising one or more nucleotides may be present in a binding complex,thus allowing the signal from the label to be detected during thepersistence time of the binding complex. One exemplary label is afluorescent label.

II. Method of Analyzing Target Nucleic Acid

Disclosed herein are multivalent binding compositions and their use inanalyzing nucleic acid including sequencing or other bioassayapplications. An increase in binding of a nucleotide to an enzyme (e.g.,polymerase) or an enzyme complex can be effected by increasing theeffective concentration of the nucleotide. The increase can be achievedby increasing the concentration of the nucleotide in free solution, orby increasing the amount of the nucleotide in proximity to the relevantbinding site. The increase can also be achieved by physicallyrestricting a number of nucleotides into a limited volume thus resultingin a local increase in concentration, and such as structure may thusbind to the binding site with a higher apparent avidity than would beobserved with unconjugated, untethered, or otherwise unrestrictedindividual nucleotide. One exemplary means of effecting such restrictionis by providing a multivalent binding composition in which multiplenucleotides are bound to a particle such as a polymer, a branchedpolymer, a dendrimer, a micelle, a liposome, a microparticle, ananoparticle, a quantum dot, or other suitable particle known in theart.

The multivalent binding composition disclosed herein can include atleast one particle-nucleotide conjugate, and the particle-nucleotideconjugate has a plurality of copies of the same nucleotide attached tothe particle. When the nucleotide is complementary to the target nucleicacid, the particle-nucleotide conjugate forms a binding complex with thepolymerase and the target nucleic acid, and the binding complex exhibitsincreased stability and longer persistence time than the binding complexformed using a single unconjugated or untethered nucleotide.

The multivalent binding composition can be used to localize detectablesignals to active regions of biochemical interactions, such as sites ofprotein-nucleic acid interactions, nucleic acid hybridization reactions,or enzymatic reactions, such as polymerase reactions. For instance, themultivalent binding composition described herein can be utilized toidentify sites of base incorporation in elongating nucleic acid chainsduring polymerase reactions and to provide base discrimination forsequencing and array based applications. The increased binding betweenthe target nucleic acid and the nucleotide in the multivalent bindingcomposition, when the nucleotide is complementary to the target nucleicacid, provides enhanced signal that greatly improve base call accuracyand shorten imaging time.

In addition, the use of multivalent binding composition allowssequencing signals from a given sequence to originate within clusterregions containing multiple copies of the target sequence. Sequencingmethods incorporating multiple copies of a target sequence have theadvantage that signals can be amplified due to the presence of multiplesimultaneous sequencing reactions within the defined region, eachproviding its own signal. The presence of multiple signals within adefined area also reduces the impact of any single skipped cycle, due tothe fact that the signal from a large number of correct base calls canoverwhelm the signal from a smaller number of skipped or incorrect basecalls, therefore providing methods for reducing phasing errors and/or toimprove read length in sequencing reactions.

The multivalent binding compositions and their use disclosed herein leadto one or more of: (i) stronger signal for better base-calling accuracycompared to conventional nucleic acid amplification and sequencingmethodologies; ii) allow greater discrimination of sequence-specificsignal from background signals; (iii) reduced requirements for theamount of starting material necessary, (iv) increased sequencing rateand shortened sequencing time; (v) reducing phasing errors, and (vi)improving read length in sequencing reactions.

In some embodiments, the target nucleic acid can refer to a targetnucleic acid sample having one or more nucleic acid molecules. In someembodiments, the target nucleic acid can include a plurality of nucleicacid molecules. In some embodiments, the target nucleic acid can includetwo or more nucleic acid molecules. In some embodiments, the targetnucleic acid can include two or more nucleic acid molecules having thesame sequences.

A. Sequencing Target Nucleic Acid

FIG. 1A-1H illustrate one exemplified method in which the multivalentbinding composition is used for sequencing a target nuclei acid. Asshown in FIG. 1A, the target nucleic acid 102 can be tethered to a solidsupport surface 101. The target nucleic acid can be attached to thesurface either directly or indirectly. Although not shown in FIG. 1A,the target nucleic acid 102 can be hybridized to an adapter, which isattached to the surface through a covalent or noncovalent bond. When oneor more adapters are used to attach the target nucleic acid to thesurface, the target surface can comprise a fragment that iscomplementary to the adapter and thus hybridize to the adaptor. In someinstances, one adapter sequence may be tethered to the surface. In someinstances, a plurality of adapter sequences may be tethered to thesurface. In some instances, the target nucleic acid 102 can also beattached directly to the solid-support surface without the use of anadapter. The solid support can be a low non-specific binding surface.

In FIG. 1B, after the initial step of attaching the target nucleic acidto the surface of a solid support surface (e.g., through hybridizationto adapters), the target nucleic acid is then clonally-amplified to formclusters of amplified nucleic acids. When the target nucleic acid isattached to the surface through an adapter, the surface density ofclonally-amplified nucleic acid sequences hybridized to adapter on thesupport surface may span the same range as the surface density oftethered primers. The clonal amplification may be performed using apolymerase chain reaction (PCR), multiple displacement amplification(MDA), transcription-mediated amplification (TMA), nucleic acidsequence-based amplification (NASBA), strand displacement amplification(SDA), real-time SDA, bridge amplification, isothermal bridgeamplification, rolling circle amplification, circle-to-circleamplification, helicase-dependent amplification, recombinase-dependentamplification, single-stranded binding (SSB) protein-dependentamplification, or any combination thereof.

FIG. 1C illustrates a non-limiting step of annealing a primer 103 to thetarget nucleic acid 102 to form a primed target nucleic acid 104. FIG.1B only shows one primer being used in the annealing step, but more thanone primers can be used depending on the types of target nucleic acid.In some instances, the adapter that is used to attach the target nucleicacid to the surface has the same sequence as the primer used to preparethe primed target nucleic acid. The primer may comprise forwardamplification primers, reverse amplification primers, sequencingprimers, and/or molecular barcoding sequences, or any combinationthereof. In some instances, one primer sequence may be used in thehybridization step. In some instances, a plurality of different primersequences may be used in the hybridization step.

As shown in FIG. 1D, the primed target nucleic acid 104 is combined witha multivalent binding composition and a polymerase 106 to form a bindingcomplex. The non-limiting example of multivalent binding composition inFIG. 1D comprises four particle-nucleotide conjugates 105 a, 105 b, 105c, and 105 d. Each particle-nucleotide conjugate has multiple copies ofa nucleotide attached to the particle, and the four particle-nucleotideconjugates cover four types of nucleotide respectively. Theparticle-nucleotide conjugate having a nucleotide that is complementaryto the next base on the target nucleic acid will form a binding complexwith the polymerase and the target nucleic acid. In some instances, themultivalent binding composition may include one, two or threeparticle-nucleotide conjugates. In some embodiments, each different typeof particle-nucleotide conjugate can be labeled with a separate label.In some embodiments, three of four types of nucleotide conjugates can belabeled, with a fourth either unlabeled or conjugated to an undetectablelabel. In some embodiments, 1, 2, 3, or 4 particle-nucleotide conjugatescan be labeled, either with the same label, or each with a labelcorresponding to the identity of its conjugated nucleotide, with,respectively, 3, 2, 1, or no particle-nucleotide conjugates that may beeither left unlabeled or conjugated to an undetectable label. In someembodiments, detection of a polymerase complex incorporating aparticle-nucleotide conjugate may be carried out using four-colordetection, such that conjugates corresponding to all four nucleotidesare present in a sample, each conjugate having a separate labelcorresponding to the nucleotide conjugated thereto. In some embodiments,the four particle-nucleotide conjugates may be exposed to or contactedwith the target nucleic acid at the same time; in some otherembodiments, the four particle-nucleotide conjugates may be exposed toor contacted with the target nucleic acid sequentially, eitherindividually, or in groups of two or three. In some embodiments,detection of a polymerase complex incorporating a particle-nucleotideconjugate may be carried out using three-color detection, such thatconjugates corresponding to three of the four nucleotides are present ina sample, with three conjugates having a separate label corresponding tothe nucleotide conjugated thereto and one conjugate having no label orbeing conjugated to an undetectable label. In some embodiments, onlythree types of conjugates are provided, such that conjugatescorresponding to three of the four nucleotides are present in a sample,with three conjugates having a separate label corresponding to thenucleotide conjugated thereto and one conjugate being absent. In someembodiments, the identity of nucleotides corresponding to an unlabeledor absent nucleotide conjugate can be determined with respect to thelocation and/or identity of “dark” spots or locations of known targetnucleic acids showing no fluorescence signal. In some embodiments, thepresent disclosure provides said method, wherein the detection of thebinding complex is performed in the absence of unbound or solution-bornepolymer nucleotide conjugates.

In some embodiments where three of the four particle-nucleotideconjugates are labeled, or where only three of the fourparticle-nucleotide conjugates are present, the identity of thenucleotide corresponding to the unlabeled or absent conjugate may beestablished by the absence of a signal or by monitoring of the presenceof unlabeled complexes such as by the identification of “dark” spots orunlabeled regions in a sequencing reaction. In some embodiments,detection of a polymerase complex incorporating a particle-nucleotideconjugate may be carried out using two-color detection, such thatconjugates corresponding to two of the four nucleotides are present in asample, with two conjugates having a separate label corresponding to thenucleotide conjugated thereto and two conjugates having no label orbeing conjugated to an undetectable label. In some embodiments, only twoof the four particle-nucleotide conjugates are labeled. In someembodiments where two of the four particle-nucleotide conjugates arelabeled, the identity of the nucleotide corresponding to the unlabeledconjugate or conjugates may be established by the absence of a signal orby monitoring of the presence of unlabeled complexes such as by theidentification of “dark” spots or unlabeled regions in a sequencingreaction. In some embodiments where two of the four particle-nucleotideconjugates are labeled, the four particle-nucleotide conjugates may beexposed to or contacted with the target nucleic acid sequentially,either individually, or in groups of two or three. In some embodimentstwo of the four particle-nucleotide conjugates may share a common label,and the four particle-nucleotide conjugates may be exposed to orcontacted with the target nucleic acid sequentially, eitherindividually, or in groups of two or three, wherein each contacting stepshows the distinction between two or more different bases, such thatafter two, three, four, or more such contacting steps the identities ofall unknown bases have been determined.

FIG. 1E shows the images captured on the surface after the bindingcomplex is formed between the polymerase, the target nucleic acid, andthe particle-nucleotide conjugate having a nucleotide commentary to thenext base of the target nucleic acid. The captured image includes fourbinding complexes 107 a, 107 b, 107 c, and 107 d formed on the surface,and each binding complex has a different nucleotide which can bedistinguished based on the label (e.g., color) on theparticle-nucleotide conjugate. Because of use of the particle-nucleotideconjugate allows binding signals from a given sequence to originatewithin cluster regions containing multiple copies of the targetsequence, the sequencing signals is greatly enhanced. Although FIG. 1Einvolves four particle-nucleotide conjugate, each having a differenttype of nucleotide, some methods can use one, two, or threeparticle-nucleotide conjugates, each having a different type ofnucleotide and label. In some embodiments, each different type ofparticle-nucleotide conjugate can be labeled either with the same label,or each with a label corresponding to the identity of its conjugatednucleotide. In some embodiments, three of four types of nucleotideconjugates can be labeled, with a fourth either unlabeled or conjugatedto an undetectable label. In some embodiments, 1, 2, 3, or 4particle-nucleotide conjugates can be labeled with a separate label,with, respectively, 3, 2, 1, or no particle-nucleotide conjugates eitherunlabeled or conjugated to an undetectable label In some embodiments, adetection step can comprise simultaneous and/or serial excitation of upto 4 different excitation wavelengths, such as wherein the fluorescenceimaging is carried out by detecting single and/or multiple fluorescenceemission bands that uniquely classify each of the possible base pairing(A,G,C, or T). In some embodiments, four different nucleic acid bindingcompositions, each comprising a different nucleotide or nucleotideanalog, may be used to determine the identity of the terminalnucleotide, wherein one of the four different nucleic acid bindingcompositions is labeled with a first fluorophore, one is labeled with asecond fluorophore, one is labeled with both the first and secondfluorophore, and one is not labeled, and wherein the detecting stepcomprises simultaneous excitation at a first excitation wavelength and asecond excitation wavelength and images are acquired at a firstfluorescence emission wavelength and a second fluorescence emissionwavelength.

When the multivalent binding composition is used in replacement ofsingle unconjugated or untethered nucleotide to form a binding complexwith the polymerase and the target nucleic acid, the local concentrationof the nucleotide is increased many fold, which in turn enhances thesignal intensity. The formed binding complex also has a longerpersistence time which in turn helps shorten the imaging step. The highsignal intensity resulted from the use of the polymer nucleotideconjugate remain for the entire binding and imaging step. The strongbinding between the polymerase, the primed target strand, and thenucleotide or nucleotide analog also means that the formed bindingcomplex will remain stable during the washing step and the signal willremain at a high intensity when other reaction mixture and unmatchednucleotide analogs are washed away. After the imaging step, the bindingcomplex can be destabilized and the primed target nucleic acid can thenbe extended for one base.

The sequencing method may further comprise incorporating the N+1 orterminal nucleotide into the primed strand as shown in FIG. 1F. In FIG.1F, the primer strand of the primed target nucleic acid 104 can beextended for one base to form an extended nucleic acid 108. Theextension step can occur after or concurrently with the destabilizationof the binding complex. The primed target nucleic acid 104 can beextended using a complementary nucleotide that is attached to theparticle in the particle-nucleotide conjugate, or using an unconjugatedor untethered free nucleotide that is provided after the multivalentbinding composition has been removed.

After the extension step, the contacting step as shown in FIG. 1G can beperformed again to form binding complexes and imitate the nextsequencing cycle. The contacting, detecting, and extension steps can berepeated for one or more cycles, thereby determining the sequence of thetarget nucleic acid molecule. For example, FIG. 1H shows the surfaceimages after multiple sequencing cycles, and the images can then beprocessed to determine the sequences of the target nucleic acidmolecules.

The extension of the primed target nucleic acid may be prevented orinhibited due to a blocked nucleotide on the strand or the use ofpolymerase that is catalytically inactive. When the nucleotide in thepolymer-nucleotide conjugate has a blocking group that prevents theextension of the nucleic acid, incorporation of a nucleotide may beachieved by the removal of a blocking group from said nucleotide (suchas by detachment of said nucleotide from its polymer, branched polymer,dendrimer, particle, or the like). When the extension of the primedtarget nucleic acid is inhibited due to the use of polymerase that iscatalytically inactive, incorporation of a nucleotide may be achieved bythe provision of a cofactor or activator such as a metal ion.

Also disclosed herein are systems configured for performing any of thedisclosed nucleic acid sequencing or nucleic acid analysis methods. Thesystem may comprise a fluid flow controller and/or fluid dispensingsystem configured to sequentially and iteratively contact the primedtarget nucleic acid molecules attached to a solid support with thedisclosed polymerase and multivalent binding compositions and/orreagents. The contacting may be performed within one or more flow cells.In some instances, said flow cells may be fixed components of thesystem. In some instances, said flow cells may be removable and/ordisposable components of the system.

The sequencing system may include an imaging module, i.e., one or morelight sources, one or more optical components, and one or more imagesensors for imaging and detection of binding of the disclosed nucleicacid binding compositions to target nucleic acid molecules tethered to asolid support or the interior of a flow cell. The disclosedcompositions, reagents, and methods may be used for any of a variety ofnucleic acid sequencing and analysis applications. Examples include, butare not limited to, DNA sequencing, RNA sequencing, whole genomesequencing, targeted sequencing, exome sequencing, genotyping, and thelike.

The sequencing system may also include computer control systems that areprogrammed to implement methods of the disclosure. The computer systemis programmed or otherwise configured to implement methods of thedisclosure including nucleic acid sequencing methods, interpretation ofnucleic acid sequencing data and analysis of cellular nucleic acids,such as RNA (e.g., mRNA), and characterization of cells from sequencingdata. The computer system can be an electronic device of a user or acomputer system that is remotely located with respect to the electronicdevice. The electronic device can be a mobile electronic device.

FIG. 3 is a flowchart outlining the steps in sequencing a target nucleicacid. 301 describes a step of attaching target library sequences to asolid support surface by hybridizing the target nucleic acid moleculesto complementary adapters on substrate surface. The target nucleic acidmolecules can be single stranded or partially double stranded. Prior to301, the nucleic acid molecules in the target library may have beenprepared to contain fragments complementary to the adaptor sequencesthrough ligation or other methods. 302 describes the step of clonalamplification to generate clusters of target nucleic acid molecules onthe surface. 303 describes hybridizing sequencing primers tocomplementary primer binding sequences on the target nucleic acid toform the primed target nucleic acid. 304 describes combining thepolymerase, the multivalent binding composition, which contains labeled(e.g., fluorescently-labeled) particle-nucleotide conjugates, and theprimed target nucleic acid. 304 may also include a step of washing orremoving the unbound reagents including polymerase andparticle-nucleotide conjugate.

In 305, when the nucleotide on the particle-nucleotide conjugate iscomplementary to the next base of the primed target nucleic acid, theparticle-nucleotide conjugate, polymerase, and primed target nucleicacid form a ternary binding complex, which can be detected by detectionmethods (e.g., florescence imaging) compatible with the label on theparticle-nucleotide conjugate. 305 can also include measuring thepersistence time of the ternary binding complex. In 306, the bindingcomplex is destabilized to remove the binding of the particle-nucleotideconjugate and polymerase. The dissociation can be achieved by placingthe binding complex in a condition (e.g., adding Strontium ions) thatwill change the conformation of the polymerase and destabilize thebinding. 306 may also include a step of washing or removing thedissociated particle-nucleotide conjugate and/or polymerase. 307describes the step of extending the primed strand of the primed targetnucleic acid by a single base addition reaction. After the single baseextension, steps 304, 305, 306, and 307 can be repeated in multiplecycles to determine the sequences of the target nucleic acid.

FIG. 4 is another flowchart outlining the steps in sequencing a targetnucleic acid, which includes cleaving a nucleotide from theparticle-nucleotide conjugate and incorporating the cleaved nucleotide.401 describes a step of attaching target library sequences to a solidsupport surface by hybridizing the target nucleic acid molecules tocomplementary adapters on substrate surface. The target nucleic acidmolecules can be single stranded or partially double stranded. Prior to401, the nucleic acid molecules in the target library may have beenprepared to contain fragments complementary to the adaptor sequencesthrough ligation or other methods. 402 describes the step of clonalamplification to generate clusters of target nucleic acid molecules onthe surface. 403 describes hybridizing sequencing primers tocomplementary primer binding sequences on the target nucleic acid toform the primed target nucleic acid. 404 describes combining thepolymerase, the multivalent binding composition, which contains labeled(e.g., fluorescently-labeled) particle-nucleotide conjugates, and theprimed target nucleic acid. In the particle-nucleotide conjugates, thenucleotides are attached to the particle through chemical bonds orinteractions that can be later severed. 404 may also include a step ofwashing or removing the unbound reagents including polymerase andparticle-nucleotide conjugate.

In 405, when the nucleotide on the particle-nucleotide conjugate iscomplementary to the next base of the primed target nucleic acid, theparticle-nucleotide conjugate, polymerase, and primed target nucleicacid form a ternary binding complex, which can be detected by detectionmethods (e.g., florescence imaging) compatible with the label on theparticle-nucleotide conjugate. 405 can also include measuring thepersistence time of the ternary binding complex. In 406, the polymeraseis placed in a condition that would make it catalytically active toincorporate a nucleotide. The condition can include exposing thepolymerase to Mg or Mn ions in the reaction solution. The nucleotidethat is bound to the polymerase and the primed target nucleic acid isthen cleaved from the particle and then incorporated into the primedstrand of the primed target nucleic acid. The binding complex isdestabilized. 406 may also include a step of washing or removing thedissociated particle-nucleotide conjugate and/or polymerase. After theextension, steps 404, 405, and 406 can be repeated in multiple cycles todetermine the sequences of the target nucleic acid.

B. Detecting Target Nucleic Acid

FIG. 4 illustrates one exemplified method in which the multivalentbinding composition is used for detecting a target nuclei acid. As shownin FIG. 4A, the polymer-nucleotide conjugate 201 is placed in contactwith polymerase 206, a first nucleic acid molecule 204 and a secondnucleic acid molecule 205. The polymer-nucleotide conjugate 201 hasmultiple polymer branches radiating from the core, and some branches areattached to nucleotide or oligonucleotide 202, and some branches areattached to a label 203. When the nucleotide or oligonucleotide 202 onthe polymer-nucleotide conjugate 201 is complementary to at least afraction of the first nucleic acid 204, a binding complex is formed asshown in FIG. 4B, and the strong binding signal can helps detect targetnucleic acid with sequences complementary or partially complementary tothe nucleotide or oligonucleotide on the polymer-nucleotide conjugate.In some instances, at least one of the polymerase, nucleic acidmolecules, and polymer-nucleotide conjugates is attached to a solidsupport.

The multivalent binding composition described herein can be used in amethod of detecting a target nucleic acid in a sample. Also disclosedherein are systems configured for performing any of the disclosednucleic acid analysis methods. The system may comprise a fluid flowcontroller and/or fluid dispensing system configured to sequentially anditeratively contact the nucleic acid molecules with the disclosedpolymerase and multivalent binding compositions and/or reagents. Thecontacting may be performed within one or more flow cells. In someinstances, said flow cells may be fixed components of the system. Insome instances, said flow cells may be removable and/or disposablecomponents of the system. The system may also include a cartridgecomprising a sample collection unit and an assay assembly, wherein thesample collection unit is configured to collect a sample, and whereinthe assay assembly comprises at least one reaction site containing amultivalent binding composition adapted to interact with said analyte,allowing the predetermined portion of sample to react with assayreagents contained within the assay assembly to yield a signalindicative of the presence of the analyte in the sample, and detectingthe signal generated from the analyte.

III. Multivalent Binding Composition

The present disclosure relates to multivalent binding compositionshaving a plurality of nucleotides conjugated to a particle (e.g., apolymer, branched polymer, dendrimer, or equivalent structure).Contacting the multivalent binding composition with a polymerase and aprimed target nucleic acid may result in the formation of a ternarycomplex which may be detected and in turn achieve a more accuratedetermination of the bases of the target nucleic acid.

When the multivalent binding composition is used in replacement ofsingle unconjugated or untethered nucleotide to form a complex with thepolymerase and the target nucleic acid, the local concentration of thenucleotide is increased many fold, which in turn enhances the signalintensity, particularly the correct signal versus mismatch. Themultivalent binding composition described herein can include at leastone particle-nucleotide conjugate for interacting with the targetnucleic acid. The multivalent composition can also include two, three,or four different particle-nucleotide conjugates, each having adifferent nucleotide conjugated to the particle.

The multivalent binding composition can comprise 1, 2, 3, 4, or moretypes of particle-nucleotide conjugates, wherein eachparticle-nucleotide conjugate comprises a different type of nucleotide.A first type of the particle-nucleotide conjugate can comprise anucleotide selected from the group consisting of ATP, ADP, AMP, dATP,dADP, and dAMP. A second type of the particle-nucleotide conjugate cancomprise a nucleotide selected from the group consisting of TTP, TDP,TMP, dTTP, dTDP, dTMP, UTP, UDP, UMP, dUTP, dUDP, and dUMP. A third typeof the particle-nucleotide conjugate can comprise a nucleotide selectedfrom the group consisting of CTP, CDP, CMP, dCTP, dCDP, and dCMP. Afourth type of the particle-nucleotide conjugate can comprise anucleotide selected from the group consisting of GTP, GDP, GMP, dGTP,dGDP, and dGMP. In some embodiments, each particle-nucleotide conjugatecomprises a single type of nucleotide respectively corresponding to oneor more nucleotide selected from the group consisting of ATP, ADP, AMP,dATP, dADP, dAMP TTP, TDP, TMP, dTTP, dTDP, dTMP, UTP, UDP, UMP, dUTP,dUDP, dUMP, CTP, CDP, CMP, dCTP, dCDP, dCMP, GTP, GDP, GMP, dGTP, dGDP,and dGMP. Each multivalent binding composition may further comprise oneor more labels corresponding to the particular nucleotide conjugated toeach respective conjugate. Exemplary labels include fluorescent labels,colorimetric labels, electrochemical labels (such as, for example,glucose or other reducing sugars, or thiols or other redox activemoieties), luminescent labels, spin labels, radioactive labels, stericlabels, affinity tags, or the like.

A. Particle-Nucleotide Conjugate

In a particle-nucleotide conjugate, multiple copies of the samenucleotide may be covalently bound to or noncovalently bound to theparticle. Examples of the particle can include a branched polymer; adendrimer; a cross linked polymer particle such as an agarose,polyacrylamide, acrylate, methacrylate, cyanoacrylate, methylmethacrylate particle; a glass particle; a ceramic particle; a metalparticle; a quantum dot; a liposome; an emulsion particle, or any otherparticle (e.g, nanoparticles, microparticles, or the like) known in theart. In a preferred embodiment, the particle is a branched polymer.

The nucleotide can be linked to the particle through a linker, and thenucleotide can be attached to one end or location of a polymer. Thenucleotide can be conjugated to the particle through the 5′ end of thenucleotide. In some particle-nucleotide conjugates, one nucleotideattached to one end or location of a polymer. In someparticle-nucleotide conjugate, multiple nucleotides are attached to oneend or location of a polymer. The conjugated nucleotide is stericallyaccessible to one or more proteins, one or more enzymes, and nucleotidebinding moieties. In some embodiments, a nucleotide may be providedseparately from a nucleotide binding moiety such as a polymerase. Insome embodiments, the linker does not comprise a photo emitting or photoabsorbing group.

The particle can also have a binding moiety. In some embodiments,particles may self-associate without the use of a separate interactionmoiety. In some embodiments, particles may self-associate due to bufferconditions or salt conditions, e.g., as in the case of calcium-mediatedinteractions of hydroxyapatite particles, lipid or polymer mediatedinteractions of micelles or liposomes, or salt-mediated aggregation ofmetallic (such as iron or gold) nanoparticles.

The particle-nucleotide conjugate can have one or more labels. Examplesof the labels include but are not limited to fluorophores, spin labels,metals or metal ions, colorimetric labels, nanoparticles, PET labels,radioactive labels, or other such label as may render said compositiondetectable by such methods as are known in the art of the detection ofmacromolecules or molecular interactions. The label may be attached tothe nucleotide (e.g. by attachment to the 5′ phosphate moiety of anucleotide), to the particle itself (e.g., to the PEG subunits), to anend of the polymer, to a central moiety, or to any other location withinsaid polymer-nucleotide conjugate which would be recognized by one ofskill in the art to be sufficient to render said composition, such as aparticle, detectable by such methods as are known in the art ordescribed elsewhere herein. In some embodiments, one or more labels areprovided so as to correspond to or differentiate a particularparticle-nucleotide conjugate.

In some embodiments, the label is a fluorophore. Exemplary fluorescentmoieties include, but are not limited to, fluorescein and fluoresceinderivatives such as carboxyfluorescein, tetrachlorofluorescein,hexachlorofluorescein, carboxynapthofluorescein, fluoresceinisothiocyanate, NHS-fluorescein, iodoacetamidofluorescein, fluoresceinmaleimide, SAMSA-fluorescein, fluorescein thiosemicarbazide,carbohydrazinomethylthioacetyl-amino fluorescein, rhodamine andrhodamine derivatives such as TRITC, TMR, lissamine rhodamine, TexasRed, rhodamine B, rhodamine 6G, rhodamine 10, NHS-rhodamine,TMR-iodoacetamide, lissamine rhodamine B sulfonyl chloride, lissaminerhodamine B sulfonyl hydrazine, Texas Red sulfonyl chloride, Texas Redhydrazide, coumarin and coumarin derivatives such as AMCA, AMCA-NHS,AMCA-sulfo-NHS, AMCA-HPDP, DCIA, AMCE-hydrazide, BODIPY and derivativessuch as BODIPY FL C3-SE, BODIPY 530/550 C3, BODIPY 530/550 C3-SE, BODIPY530/550 C3 hydrazide, BODIPY 493/503 C3 hydrazide, BODIPY FL C3hydrazide, BODIPY FL IA, BODIPY 530/551 IA, Br-BODIPY 493/503, CascadeBlue and derivatives such as Cascade Blue acetyl azide, Cascade Bluecadaverine, Cascade Blue ethylenediamine, Cascade Blue hydrazide,Lucifer Yellow and derivatives such as Lucifer Yellow iodoacetamide,Lucifer Yellow CH, cyanine and derivatives such as indolium basedcyanine dyes, benzo-indolium based cyanine dyes, pyridium based cyaninedyes, thiozolium based cyanine dyes, quinolinium based cyanine dyes,imidazolium based cyanine dyes, Cy 3, Cy5, lanthanide chelates andderivatives such as BCPDA, TBP, TMT, BHHCT, BCOT, Europium chelates,Terbium chelates, Alexa Fluor dyes, DyLight dyes, Atto dyes, LightCyclerRed dyes, CAL Flour dyes, JOE and derivatives thereof, Oregon Greendyes, WellRED dyes, IRD dyes, phycoerythrin and phycobilin dyes,Malachite green, stilbene, DEG dyes, NR dyes, near-infrared dyes andothers known in the art such as those described in Haugland, MolecularProbes Handbook, (Eugene, Oreg.) 6th Edition; Lakowicz, Principles ofFluorescence Spectroscopy, 2nd Ed., Plenum Press New York (1999), orHermanson, Bioconjugate Techniques, 2nd Edition, or derivatives thereof,or any combination thereof. Cyanine dyes may exist in either sulfonatedor non-sulfonated forms, and consist of two indolenin, benzo-indolium,pyridium, thiozolium, and/or quinolinium groups separated by apolymethine bridge between two nitrogen atoms. Commercially availablecyanine fluorophores include, for example, Cy3, (which may comprise1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indoliumor1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-3,3-dimethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium-5-sulfonate),Cy5 (which may comprise1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-(2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-indolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indol-1-iumor1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-sulfoindolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indol-1-ium-5-sulfonate),and Cy7 (which may comprise1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-1,3-dihydro-2H-indol-2-ylidene)hepta-1,3,5-trien-1-yl]-3H-indoliumor1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene)hepta-1,3,5-trien-1-yl]-3H-indolium-5-sulfonate),where “Cy” stands for ‘cyanine’, and the first digit identifies thenumber of carbon atoms between two indolenine groups. Cy2 which is anoxazole derivative rather than indolenin, and the benzo-derivatizedCy3.5, Cy5.5 and Cy7.5 are exceptions to this rule.

In some embodiments, the detection label can be a FRET pair, such thatmultiple classifications can be performed under a single excitation andimaging step. As used herein, FRET may comprise excitation exchange(Forster) transfers, or electron-exchange (Dexter) transfers.

B. Polymer-Nucleotide Conjugate

One example of the particle-nucleotide conjugate is a polymer-nucleotideconjugate. Some non-limiting examples of the polymer-nucleotideconjugates are shown in FIG. 5A-5C. For example, FIG. 5A showspolymer-nucleotide conjugates having various configurations; FIG. 5Bshows a polymer-nucleotide conjugate having the polymer branch radiatingfrom the center; and FIG. 5C shows polymer-nucleotide conjugates havinga binding moiety such as a biotin.

Examples of the branched polymer include polyethylene glycol (PEG),polypropylene glycol, polyvinyl alcohol, polylactic acid, polyglycolicacid, polyglycine, polyvinyl acetate, a dextran, or other such polymers,or copolymers incorporating any two or more of the foregoing orincorporating other polymers as are known in the art. In one embodiment,the polymer is a PEG. In another embodiment, the polymer can have PEGbranches.

Suitable polymers may be characterized by a repeating unit incorporatinga functional group suitable for derivatization such as an amine, ahydroxyl, a carbonyl, or an allyl group. The polymer can also have oneor more pre-derivatized substituents such that one or more particularsubunits will incorporate a site of derivatization or a branch site,whether or not other subunits incorporate the same site, substituent, ormoiety. A pre-derivatized substituent may comprise or may furthercomprise, for example, a nucleotide, a nucleoside, a nucleotide analog,a label such as a fluorescent label, radioactive label, or spin label,an interaction moiety, an additional polymer moiety, or the like, or anycombination of the foregoing.

In the polymer-nucleotide conjugate, the polymer can have a plurality ofbranches. The branched polymer can have various configurations,including but are not limited to stellate (“starburst”) forms,aggregated stellate (“helter skelter”) forms, bottle brush, ordendrimer. The branched polymer can radiate from a central attachmentpoint or central moiety, or may incorporate multiple branch points, suchas, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more branch points. Insome embodiments, each subunit of a polymer may optionally constitute aseparate branch point.

The length and size of the branch can differ based on the type ofpolymer. In some branched polymers, the branch may have a length ofbetween 1 and 1,000 nm, between 1 and 100 nm, between 1 and 200 nm,between 1 and 300 nm, between 1 and 400 nm, between 1 and 500 nm,between 1 and 600 nm, between 1 and 700 nm, between 1 and 800 nm, orbetween 1 and 900 nm, or more, or having a length falling within orbetween any of the values disclosed herein. In some branched polymers,the branch may have a size corresponding to an apparent molecular weightof 1K, 2K, 3K, 4K, 5K, 10K, 15K, 20K, 30K, 50K, 80K, 100K, or any valuewithin a range defined by any two of the foregoing. The apparentmolecular weight of a polymer may be calculated from the known molecularweight of a representative number of subunits, as determined by sizeexclusion chromatography, as determined by mass spectrometry, or asdetermined by any other method as is known in the art. The polymer canhave multiple branches. The number of branches in the polymer can be 2,3, 4, 5, 6, 7, 8, 12, 16, 24, 32, 64, 128 or more, or a number fallingwithin a range defined by any two of these values.

For the polymer-nucleotide conjugate, the branched polymer of 4, 8, 16,32, or 64 branches can have nucleotides attached to the ends of PEGbranches, such that each end has attached thereto 0, 1, 2, 3, 4, 5, 6 ormore nucleotides. In one non-limiting example, the branched polymer ofbetween 3 and 128 PEG arms having attached to the polymer branches endsone or more nucleotides, such that each end has attached thereto 0, 1,2, 3, 4, 5, 6 or more nucleotides or nucleotide analogs. In someembodiments, a branched polymer or dendrimer has an even number of arms.In some embodiments, a branched polymer or dendrimer has an odd numberof arms.

In the polymer-nucleotide conjugate, each branch or a subset of branchesof the polymer may have attached thereto a moiety comprising anucleotide (e.g., an adenine, a thymine, a uracil, a cytosine, or aguanine residue or a derivative or mimetic thereof), and the moiety iscapable of binding to a polymerase, reverse transcriptase, or othernucleotide binding domain. Optionally, the moiety may be capable ofbeing incorporated into an elongating nucleic acid chain during apolymerase reaction. In some instances, said moiety may be blocked suchthat it is not capable of being incorporated into an elongating nucleicacid chain during a polymerase reaction. In some other instances, saidmoiety may be reversibly blocked such that it is not capable of beingincorporated into an elongating nucleic acid chain during a polymerasereaction until such block is removed, after which said moiety is thencapable of being incorporated into an elongating nucleic acid chainduring a polymerase reaction.

The nucleotide can be conjugated to the polymer branch through the 5′end of the nucleotide. In some instances, the nucleotide may be modifiedso as to inhibit or prevent incorporation of the nucleotide into anelongating nucleic acid chain during a polymerase reaction. By way ofexample, the nucleotide may include a 3′ deoxyribonucleotide, a 3′azidonucleotide, a 3′-methyl azido nucleotide, or another suchnucleotide as is or may be known in the art, so as to not be capable ofbeing incorporated into an elongating nucleic acid chain during apolymerase reaction. In some embodiments, the nucleotide can include a3′-O-azido group, a 3′-O-azidomethyl group, a 3′-phosphorothioate group,a 3′-O-malonyl group, a 3′-O-alkyl hydroxylamino group, or a 3′-O-benzylgroup. In some embodiments, the nucleotide lacks a 3′ hydroxyl group.

The polymer can further have a binding moiety in each branch or a subsetof branches. Some examples of the binding moiety include but are notlimited to biotin, avidin, strepavidin or the like, polyhistidinedomains, complementary paired nucleic acid domains, G-quartet formingnucleic acid domains, calmodulin, maltose-binding protein, cellulase,maltose, sucrose, glutathione-S-transferase, glutathione,O-6-methylguanine-DNA methyltransferase, benzylguanine and derivativesthereof, benzylcysteine and derivatives thereof, an antibody, anepitope, a protein A, a protein G. The binding moiety can be anyinteractive molecules or fragment thereof known in the art to bind to orfacilitate interactions between proteins, between proteins and ligands,between proteins and nucleic acids, between nucleic acids, or betweensmall molecule interaction domains or moieties.

In some embodiments, a composition as provided herein may comprise oneor more elements of a complementary interaction moiety. Exemplarycomplementary interaction moieties include, for example, biotin andavidin; SNAP-benzylguanosine; antibody or FAB and epitope; IgG FC andProtein A, Protein G, Protein A/G, or Protein L; maltose binding proteinand maltose; lectin and cognate polysaccharide; ion chelation moieties,complementary nucleic acids, nucleic acids capable of forming triplex ortriple helical interactions; nucleic acids capable of formingG-quartets, and the like. One of skill in the art will readily recognizethat many pairs of moieties exist and are commonly used for theirproperty of interacting strongly and specifically with one another; andthus any such complementary pair or set is considered to be suitable forthis purpose in constructing or envisioning the compositions of thepresent disclosure. In some embodiments, a composition as disclosedherein may comprise compositions in which one element of a complementaryinteraction moiety is attached to one molecule or multivalent ligand,and the other element of the complementary interaction moiety isattached to a separate molecule or multivalent ligand. In someembodiments, a composition as disclosed herein may comprise compositionsin which both or all elements of a complementary interaction moiety areattached to a single molecule or multivalent ligand. In someembodiments, a composition as disclosed herein may comprise compositionsin which both or all elements of a complementary interaction moiety areattached to separate arms of, or locations on, a single molecule ormultivalent ligand. In some embodiments, a composition as disclosedherein may comprise compositions in which both or all elements of acomplementary interaction moiety are attached to the same arm of, orlocations on, a single molecule or multivalent ligand. In someembodiments, compositions comprising one element of a complementaryinteraction moiety and compositions comprising another element of acomplementary interaction moiety may be simultaneously or sequentiallymixed. In some embodiments, interactions between molecules or particlesas disclosed herein allow for the association or aggregation of multiplemolecules or particles such that, for example, detectable signals areincreased. In some embodiments, fluorescent, colorimetric, orradioactive signals are enhanced. In other embodiments, otherinteraction moieties as disclosed herein or as are known in the art arecontemplated. In some embodiments, a composition as provided herein maybe provided such that one or more molecules comprising a firstinteraction moiety such as, for example, one or more imidazole orpyridine moieties, and one or more additional molecules comprising asecond interaction moiety such as, for example, histidine residues, aresimultaneously or sequentially mixed. In some embodiments, saidcomposition comprises 1, 2, 3, 4, 5, 6, or more imidazole or pyridinemoieties. In some embodiments, said composition comprises 1, 2, 3, 4, 5,6, or more histidine residues. In such embodiments, interaction betweenthe molecules or particles as provided may be facilitated by thepresence of a divalent cation such as nickel, manganese, magnesium,calcium, strontium, or the like. In some embodiments, for example, a(His)₃ group may interact with a (His)₃ group on another molecule orparticle via coordination of a nickel or manganese ion.

The multivalent binding composition may comprise one or more buffers,salts, ions, or additives. In some embodiments, representative additivesmay include, but are not limited to, betaine, spermidine, detergentssuch as Triton X-100, Tween 20, SDS, or NP-40, ethylene glycol,polyethylene glycol, dextran, polyvinyl alcohol, vinyl alcohol,methylcellulose, heparin, heparan sulfate, glycerol, sucrose,1,2-propanediol, DMSO, N,N,N-trimethylglycine, ethanol, ethoxyethanol,propylene glycol, polypropylene glycol, block copolymers such as thePluronic (r) series polymers, arginine, histidine, imidazole, or anycombination thereof, or any substance known in the art as a DNA“relaxer” (a compound, with the effect of altering the persistencelength of DNA, altering the number of within-polymer junctions orcrossings, or altering the conformational dynamics of a DNA moleculesuch that the accessibility of sites within the strand to DNA bindingmoieties is increased).

The multivalent binding composition may include zwitterionic compoundsas additives. Further representative additives may be found in Lorenz,T. C. J. Vis. Exp. (63), e3998, doi:10.3791/3998 (2012), which is herebyincorporated by reference with respect to its disclosure of additivesfor the facilitation of nucleic acid binding or dynamics, or thefacilitation of processes involving the manipulation, use, or storage ofnucleic acids. In some embodiments, representative cations may include,but are not limited to, sodium, magnesium, strontium, potassium,manganese, calcium, lithium, nickel, cobalt, or other such cations asare known in the art to facilitate nucleic acid interactions, such asself-association, secondary or tertiary structure formation, basepairing, surface association, peptide association, protein binding, orthe like.

IV. Binding Between Target Nucleic Acid and Multivalent BindingComposition

When the multivalent binding composition is used in replacement ofsingle unconjugated or untethered nucleotide to form a complex with thepolymerase and the target nucleic acid, the local concentration of thenucleotide is increased many folds, which in turn enhances the signalintensity, particularly the correct signal versus mismatch. The presentdisclosure contemplates contacting the multivalent binding compositionwith a polymerase and a primed target nucleic acid to determine theformation of a ternary binding complex.

FIG. 6 has demonstrated the use of the polymer-nucleotide conjugateincreased the signal intensity during binding, persistence, andwashing/removal steps. Because of the increased local concentration ofthe nucleotide on the polymer-nucleotide conjugate, the binding betweenthe polymerase, the primed target strand, and the nucleotide, when thenucleotide is complementary to the next base of the target nucleic acid,becomes more favorable. The formed binding complex has a longerpersistence time which in turn helps shorten the imaging step. The highsignal intensity resulted from the use of the polymer nucleotideconjugate remain for the entire binding and imaging step. The strongbinding between the polymerase, the primed target strand, and thenucleotide or nucleotide analog also means that the formed bindingcomplex will remain stabilized during the washing step and the signalwill remain at a high intensity when other reaction mixture andunmatched nucleotide analogs are washed away. After the imaging step,the binding complex can be destabilized and the primed target nucleicacid can then be extended for one base. After the extension, the bindingand imaging steps can be repeated again with the use of the polymernucleotide conjugate to determine the identity of the next base.

As an example, a graphical depiction of the increase in signal intensityduring binding, persistence, and washing/removal of a multivalentsubstrate as described herein is provided in FIG. 10, which isrepresentative of the changes in signal intensity that have beenobserved experimentally. Therefore, the compositions and methods of thepresent disclosure provide a robust and controllable means ofestablishing and maintaining a ternary enzyme complex, as well asproviding vastly improved means by which the presence of said complexmay be identified and/or measured, and a means by which the persistenceof said complex may be controlled. This provides important solutions toproblems such as that of determining the identity of the N+1 base innucleic acid sequencing applications.

Without intending to be bound by any particular theory, it has beenobserved that multivalent binding compositions disclosed hereinassociate with polymerase nucleotide complexes in order to form aternary binding complexes with a rate that is time-dependent, thoughsubstantially slower than the rate of association known to be obtainableby nucleotides in free solution. Thus, the on-rate (Kon) issubstantially and surprisingly slower than the on rate for singlenucleotides or nucleotides not attached to multivalent ligand complexes.Importantly, however, the off rate (Koff) of the multivalent ligandcomplex is substantially slower than that observed for nucleotides infree solution. Therefore, the multivalent ligand complexes of thepresent disclosure provide a surprising and beneficial improvement ofthe persistence of ternary polymerase-polynucleotide-nucleotidecomplexes (especially over such complexes that are formed with freenucleotides) allowing, for example, significant improvements in imagingquality for nucleic acid sequencing applications, over currentlyavailable methods and reagents. Importantly, this property of themultivalent substrates disclosed herein renders the formation of visibleternary complexes controllable, such that subsequent visualization,modification, or processing steps may be undertaken essentially withoutregard to the dissociation of the complex—that is, the complex can beformed, imaged, modified, or used in other ways as necessary, and willremain stable until a user carries out an affirmative dissociation step,such as exposing the complexes to a dissociation buffer.

In various embodiments, polymerases suitable for the binding interactiondescribe herein include may include any polymerase as is or may be knownin the art. It is, for example, known that every organism encodes withinits genome one or more DNA polymerases. Exemplary polymerases mayinclude but are not limited to: Klenow DNA polymerase, Thermus aquaticusDNA polymerase I (Taq polymerase), KlenTaq polymerase, and bacteriophageT7 DNA polymerase; human alpha, delta and epsilon DNA polymerases;bacteriophage polymerases such as T4, RB69 and phi29 bacteriophage DNApolymerases, Pyrococcus furiosus DNA polymerase (Pfu polymerase);Bacillus subtilis DNA polymerase III, and E. coli DNA polymerase IIIalpha and epsilon; 9 degree N polymerase, reverse transcriptases such asHIV type M or O reverse transcriptases, avian myeloblastosis virusreverse transcriptase, or Moloney Murine Leukemia Virus (MMLV) reversetranscriptase, or telomerase. Further non-limiting examples of DNApolymerases can include those from various Archaea genera, such as,Aeropyrum, Archaeglobus, Desulfurococcus, Pyrobaculum, Pyrococcus,Pyrolobus, Pyrodictium, Staphylothermus, Stetteria, Sulfolobus,Thermococcus, and Vulcanisaeta and the like or variants thereof,including such polymerases as are known in the art such as Vent™, DeepVent™, Pfu, KOD, Pfx, Therminator™, and Tgo polymerases. In someembodiments, the polymerase is a klenow polymerase.

The ternary complex has longer persistence time when the nucleotide onthe polymer-nucleotide conjugate is complementary to the target nucleicacid than when a non-complementary nucleotide. The ternary complex alsohas longer persistence time when the nucleotide on thepolymer-nucleotide conjugate is complementary to the target nucleic acidthan a complementary nucleotide that is not conjugated or tethered. Forexample, in some embodiments, said ternary complexes may have apersistence time of less than 1 s, greater than 1 s, greater than 2 s,greater than 3 s, greater than 5 s, greater than 10 s, greater than 15s, greater than 20 s, greater than 30 s, greater than 60 s, greater than120 s, greater than 360 s, greater than 3600 s, or more, or for a timelying within a range defined by any two or more of these values.

The persistence time can be measured, for example, by observing theonset and/or duration of a binding complex, such as by observing asignal from a labeled component of the binding complex. For example, alabeled nucleotide or a labeled reagent comprising one or morenucleotides may be present in a binding complex, thus allowing thesignal from the label to be detected during the persistence time of thebinding complex.

It has been observed that different ranges of persistence times areachievable with different salts or ions, showing, for example, thatcomplexes formed in the presence of, for example, Mg form more quicklythan complexes formed with other ions. It has also been observed thatcomplexes formed in the presence of, for example, Sr, form readily anddissociate completely or with substantial completeness upon withdrawalof the ion or upon washing with buffer lacking one or more components ofthe present compositions, such as, e.g., a polymer and/or one or morenucleotides, and/or one or more interaction moieties, or a buffercontaining, for example, a chelating agent which may cause or acceleratethe removal of a divalent cation from the multivalent reagent containingcomplex. Thus, in some embodiments, a composition of the presentdisclosure comprises Mg. In some embodiments, a composition of thepresent disclosure comprises Ca. In some embodiments, a composition ofthe present disclosure comprises Sr. In some embodiments, a compositionof the present disclosure comprises Co. In some embodiments, acomposition of the present disclosure comprises MgCl₂. In someembodiments, a composition of the present disclosure comprises CaCl₂. Insome embodiments, a composition of the present disclosure comprisesSrCl₂. In some embodiments, a composition of the present disclosurecomprises Co Cl₂. In some embodiments, the composition comprises no, orsubstantially no Magnesium. In some embodiments, the compositioncomprises no, or substantially no Calcium. In some embodiments, themethods of the present disclosure provide for the contacting of one ormore nucleic acids with one or more of the compositions disclosed hereinwherein said composition lacks either one of calcium or magnesium, orlacks both calcium or magnesium.

The dissociation of ternary complexes can be controlled by changing thebuffer conditions. After the imaging step, a buffer with increased saltcontent is used to cause dissociation of the ternary complexes such thatlabeled polymer-nucleotide conjugates can be washed out, providing ameans by which signals can be attenuated or terminated, such as in thetransition between one sequencing cycle and the next. This dissociationmay be effected, in some embodiments, by washing the complexes with abuffer lacking a necessary metal or cofactor. In some embodiments, awash buffer may comprise one or more compositions for the purpose ofmaintaining pH control. In some embodiments, a wash buffer may compriseone or more monovalent cations, such as sodium. In some embodiments, awash buffer lacks or substantially lacks a divalent cation, for example,having no or substantially no strontium, calcium, magnesium, ormanganese. In some embodiments, a wash buffer further comprises achelating agent, such as, for example, EDTA, EGTA, nitrilotriaceticacid, polyhistidine, imidazole, or the like. In some embodiments, a washbuffer may maintain the pH of the environment at the same level as forthe bound complex. In some embodiments, a wash buffer may raise or lowerthe pH of the environment relative to the level seen for the boundcomplex. In some embodiments, the pH may be within a range from 2-4,2-7, 5-8, 7-9, 7-10, or lower than 2, or higher than 10, or a rangedefined by any two of the values provided herein.

Addition of a particular ion may affect the binding of the polymerase toa primed target nucleic acid, the formation of a ternary complex, thedissociation of a ternary complex, or the incorporation of one or morenucleotides into an elongating nucleic acid such as during a polymerasereaction. In some embodiments, relevant anions may comprise chloride,acetate, gluconate, sulfate, phosphate, or the like. In someembodiments, an ion may be incorporated into the compositions of thepresent disclosure by the addition of one or more acids, bases, orsalts, such as NiCl₂, CoCl₂, MgCl₂, MnCl₂, SrCl₂, CaCl₂, CaSO₄, SrCO₃,BaCl₂ or the like. Representative salts, ions, solutions and conditionsmay be found in Remington: The Science and Practice of Pharmacy, 20th.Edition, Gennaro, A. R., Ed. (2000), which is hereby incorporated byreference in its entirety, and especially with respect to Chapter 17 andrelated disclosure of salts, ions, salt solutions, and ionic solutions.

The present disclosure contemplates contacting the multivalent bindingcomposition comprising at least one particle-nucleotide conjugate withone or more polymerases. The contacting can be optionally done in thepresence of one or more target nucleic acids. In some embodiments, saidtarget nucleic acids are single stranded nucleic acids. In someembodiments, said target nucleic acids are primed single strandednucleic acids. In some embodiments, said target nucleic acids are doublestranded nucleic acids. In some embodiments, said contacting comprisescontacting the multivalent binding composition with one polymerase. Insome embodiments, said contacting comprises the contacting of saidcomposition comprising one or more nucleotides with multiplepolymerases. The polymerase can be bound to a single nucleic acidmolecule.

The binding between target nucleic acid and multivalent bindingcomposition may be provided in the presence of a polymerase that hasbeen rendered catalytically inactive. In one embodiment, the polymerasemay have been rendered catalytically inactive by mutation. In oneembodiment, the polymerase may have been rendered catalytically inactiveby chemical modification. In some embodiments, the polymerase may havebeen rendered catalytically inactive by the absence of a necessarysubstrate, ion, or cofactor. In some embodiments, the polymerase enzymemay have been rendered catalytically inactive by the absence ofmagnesium ions.

The binding between target nucleic acid and multivalent bindingcomposition occur in the presence of a polymerase wherein the bindingsolution, reaction solution, or buffer lacks magnesium or manganese.Alternatively, the binding between target nucleic acid and multivalentbinding composition occur in the presence of a polymerase wherein thebinding solution, reaction solution, or buffer comprises calcium orstrontium.

When the catalytically inactive polymerases are used to help a nucleicacid interact with a multivalent binding composition, the interactionbetween said composition and said polymerase stabilizes a ternarycomplex so as to render the complex detectable by fluorescence or byother methods as disclosed herein or otherwise known in the art. Unboundpolymer-nucleotide conjugates may optionally be washed away prior todetection of the ternary binding complex.

Contacting of one or more nucleic acids with the polymer-nucleotideconjugates disclosed herein in a solution containing either one ofcalcium or magnesium, or containing both calcium and magnesium.Alternatively, the contacting of one or more nucleic acids with thepolymer-nucleotide conjugates disclosed herein in a solution lackingeither one of calcium or magnesium, or lacking both calcium ormagnesium, and in a separate step, without regard to the order of thesteps, adding to the solution one of calcium or magnesium, or bothcalcium and magnesium. In some embodiments, the contacting of one ormore nucleic acids with the polymer-nucleotide conjugates disclosedherein in a solution lacking strontium, and comprises in a separatestep, without regard to the order of the steps, adding to the solutionstrontium.

V. Use of Multivalent Binding Composition in Combination with LowNon-Specific Binding Surface

Disclosed herein are solid supports comprising low non-specific bindingsurface compositions that enable improved nucleic acid hybridization andamplification performance. In general, the disclosed supports maycomprise a substrate (or support structure), one or more layers of acovalently or non-covalently attached low-binding, chemical modificationlayers, e.g., silane layers, polymer films, and one or more covalentlyor non-covalently attached primer sequences that may be used fortethering single-stranded target nucleic acid(s) to the support surface.In some instances, the formulation of the surface, e.g., the chemicalcomposition of one or more layers, the coupling chemistry used tocross-link the one or more layers to the support surface and/or to eachother, and the total number of layers, may be varied such thatnon-specific binding of proteins, nucleic acid molecules, and otherhybridization and amplification reaction components to the supportsurface is minimized or reduced relative to a comparable monolayer.Often, the formulation of the surface may be varied such thatnon-specific hybridization on the support surface is minimized orreduced relative to a comparable monolayer. The formulation of thesurface may be varied such that non-specific amplification on thesupport surface is minimized or reduced relative to a comparablemonolayer. The formulation of the surface may be varied such thatspecific amplification rates and/or yields on the support surface aremaximized. Amplification levels suitable for detection are achieved inno more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or more than 30amplification cycles in some cases disclosed herein.

Examples of materials from which the substrate or support structure maybe fabricated include, but are not limited to, glass, fused-silica,silicon, a polymer (e.g., polystyrene (PS), macroporous polystyrene(MPPS), polymethylmethacrylate (PMMA), polycarbonate (PC), polypropylene(PP), polyethylene (PE), high density polyethylene (HDPE), cyclic olefinpolymers (COP), cyclic olefin copolymers (COC), polyethyleneterephthalate (PET)), or any combination thereof. Various compositionsof both glass and plastic substrates are contemplated.

The substrate or support structure may be rendered in any of a varietyof geometries and dimensions known to those of skill in the art, and maycomprise any of a variety of materials known to those of skill in theart. For example, in some instances the substrate or support structuremay be locally planar (e.g., comprising a microscope slide or thesurface of a microscope slide). Globally, the substrate or supportstructure may be cylindrical (e.g., comprising a capillary or theinterior surface of a capillary), spherical (e.g., comprising the outersurface of a non-porous bead), or irregular (e.g., comprising the outersurface of an irregularly-shaped, non-porous bead or particle). In someinstances, the surface of the substrate or support structure used fornucleic acid hybridization and amplification may be a solid, non-poroussurface. In some instances, the surface of the substrate or supportstructure used for nucleic acid hybridization and amplification may beporous, such that the coatings described herein penetrate the poroussurface, and nucleic acid hybridization and amplification reactionsperformed thereon may occur within the pores.

The substrate or support structure that comprises the one or morechemically-modified layers, e.g., layers of a low non-specific bindingpolymer, may be independent or integrated into another structure orassembly. For example, in some instances, the substrate or supportstructure may comprise one or more surfaces within an integrated orassembled microfluidic flow cell. The substrate or support structure maycomprise one or more surfaces within a microplate format, e.g., thebottom surface of the wells in a microplate. As noted above, in somepreferred embodiments, the substrate or support structure comprises theinterior surface (such as the lumen surface) of a capillary. Inalternate preferred embodiments the substrate or support structurecomprises the interior surface (such as the lumen surface) of acapillary etched into a planar chip.

As noted, the low non-specific binding supports of the presentdisclosure exhibit reduced non-specific binding of proteins, nucleicacids, and other components of the hybridization and/or amplificationformulation used for solid-phase nucleic acid amplification. The degreeof non-specific binding exhibited by a given support surface may beassessed either qualitatively or quantitatively. For example, in someinstances, exposure of the surface to fluorescent dyes (e.g., cyaninssuch as Cy3, or Cy5, etc., fluoresceins, coumarins, rhodamines, etc. orother dyes disclosed herein), fluorescently-labeled nucleotides,fluorescently-labeled oligonucleotides, and/or fluorescently-labeledproteins (e.g. polymerases) under a standardized set of conditions,followed by a specified rinse protocol and fluorescence imaging may beused as a qualitative tool for comparison of non-specific binding onsupports comprising different surface formulations. In some instances,exposure of the surface to fluorescent dyes, fluorescently-labelednucleotides, fluorescently-labeled oligonucleotides, and/orfluorescently-labeled proteins (e.g. polymerases) under a standardizedset of conditions, followed by a specified rinse protocol andfluorescence imaging may be used as a quantitative tool for comparisonof non-specific binding on supports comprising different surfaceformulations—provided that care has been taken to ensure that thefluorescence imaging is performed under conditions where fluorescencesignal is linearly related (or related in a predictable manner) to thenumber of fluorophores on the support surface (e.g., under conditionswhere signal saturation and/or self-quenching of the fluorophore is notan issue) and suitable calibration standards are used. In someinstances, other techniques known to those of skill in the art, forexample, radioisotope labeling and counting methods may be used forquantitative assessment of the degree to which non-specific binding isexhibited by the different support surface formulations of the presentdisclosure.

Some surfaces disclosed herein exhibit a ratio of specific tononspecific binding of a fluorophore such as Cy3 of at least 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40,50, 75, 100, or greater than 100, or any intermediate value spanned bythe range herein. Some surfaces disclosed herein exhibit a ratio ofspecific to nonspecific fluorescence of a fluorophore such as Cy3 of atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or anyintermediate value spanned by the range herein.

As noted, in some instances, the degree of non-specific bindingexhibited by the disclosed low-binding supports may be assessed using astandardized protocol for contacting the surface with a labeled protein(e.g., bovine serum albumin (BSA), streptavidin, a DNA polymerase, areverse transcriptase, a helicase, a single-stranded binding protein(SSB), etc., or any combination thereof), a labeled nucleotide, alabeled oligonucleotide, etc., under a standardized set of incubationand rinse conditions, followed be detection of the amount of labelremaining on the surface and comparison of the signal resultingtherefrom to an appropriate calibration standard. In some instances, thelabel may comprise a fluorescent label. In some instances, the label maycomprise a radioisotope. In some instances, the label may comprise anyother detectable label known to one of skill in the art. In someinstances, the degree of non-specific binding exhibited by a givensupport surface formulation may thus be assessed in terms of the numberof non-specifically bound protein molecules (or other molecules) perunit area. In some instances, the low-binding supports of the presentdisclosure may exhibit non-specific protein binding (or non-specificbinding of other specified molecules, (e.g., cyanins such as Cy3, orCy5, etc., fluoresceins, coumarins, rhodamines, etc. or other dyesdisclosed herein)) of less than 0.001 molecule per μm², less than 0.01molecule per μm², less than 0.1 molecule per μm², less than 0.25molecule per μm², less than 0.5 molecule per μm², less than 1 moleculeper μm², less than 10 molecules per μm², less than 100 molecules perμm², or less than 1,000 molecules per μm². Those of skill in the artwill realize that a given support surface of the present disclosure mayexhibit non-specific binding falling anywhere within this range, forexample, of less than 86 molecules per μm². For example, some modifiedsurfaces disclosed herein exhibit nonspecific protein binding of lessthan 0.5 molecule/μm² following contact with a 1 μM solution of Cy3labeled streptavidin (GE Amersham) in phosphate buffered saline (PBS)buffer for 15 minutes, followed by 3 rinses with deionized water. Somemodified surfaces disclosed herein exhibit nonspecific binding of Cy3dye molecules of less than 0.25 molecules per μm². In independentnonspecific binding assays, 1 μM labeled Cy3 SA (ThermoFisher), 1 μM Cy5SA dye (ThermoFisher), 10 μM Aminoallyl-dUTP-ATTO-647N (JenaBiosciences), 10 μM Aminoallyl-dUTP-ATTO-Rho11 (Jena Biosciences), 10 μMAminoallyl-dUTP-ATTO-Rho11 (Jena Biosciences), 10 μM7-Propargylamino-7-deaza-dGTP-Cy5 (Jena Biosciences, and 10 μM7-Propargylamino-7-deaza-dGTP-Cy3 (Jena Biosciences) were incubated onthe low binding substrates at 37° C. for 15 minutes in a 384 well plateformat. Each well was rinsed 2-3× with 50 ul deionized RNase/DNase Freewater and 2-3× with 25 mM ACES buffer pH 7.4. The 384 well plates wereimaged on a GE Typhoon instrument using the Cy3, AF555, or Cy5 filtersets (according to dye test performed) as specified by the manufacturerat a PMT gain setting of 800 and resolution of 50-100 μm. For higherresolution imaging, images were collected on an Olympus IX83 microscope(Olympus Corp., Center Valley, Pa.) with a total internal reflectancefluorescence (TIRF) objective (100×, 1.5 NA, Olympus), a CCD camera(e.g., an Olympus EM-CCD monochrome camera, Olympus XM-10 monochromecamera, or an Olympus DP80 color and monochrome camera), an illuminationsource (e.g., an Olympus 100 W Hg lamp, an Olympus 75 W Xe lamp, or anOlympus U-HGLGPS fluorescence light source), and excitation wavelengthsof 532 nm or 635 nm. Dichroic mirrors were purchased from Semrock (IDEXHealth & Science, LLC, Rochester, N.Y.), e.g., 405, 488, 532, or 633 nmdichroic reflectors/beamsplitters, and band pass filters were chosen as532 LP or 645 LP concordant with the appropriate excitation wavelength.Some modified surfaces disclosed herein exhibit nonspecific binding ofdye molecules of less than 0.25 molecules per μm².

In some instances, the surfaces disclosed herein exhibit a ratio ofspecific to nonspecific binding of a fluorophore such as Cy3 of at least2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate valuespanned by the range herein. In some instances, the surfaces disclosedherein exhibit a ratio of specific to nonspecific fluorescence signalsfor a fluorophore such as Cy3 of at least 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, orgreater than 100, or any intermediate value spanned by the range herein.

The low-background surfaces consistent with the disclosure herein mayexhibit specific dye attachment (e.g., Cy3 attachment) to non-specificdye adsorption (e.g., Cy3 dye adsorption) ratios of at least 4:1, 5:1,6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1, or more than 50specific dye molecules attached per molecule nonspecifically adsorbed.Similarly, when subjected to an excitation energy, low-backgroundsurfaces consistent with the disclosure herein to which fluorophores,e.g., Cy3, have been attached may exhibit ratios of specificfluorescence signal (e.g., arising from Cy3-labeled oligonucleotidesattached to the surface) to non-specific adsorbed dye fluorescencesignals of at least 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1,30:1, 40:1, 50:1, or more than 50:1.

In some instances, the degree of hydrophilicity (or “wettability” withaqueous solutions) of the disclosed support surfaces may be assessed,for example, through the measurement of water contact angles in which asmall droplet of water is placed on the surface and its angle of contactwith the surface is measured using, e.g., an optical tensiometer. Insome instances, a static contact angle may be determined. In someinstances, an advancing or receding contact angle may be determined. Insome instances, the water contact angle for the hydrophilic, low-bindingsupport surfaced disclosed herein may range from about 0 degrees toabout 30 degrees. In some instances, the water contact angle for thehydrophilic, low-binding support surfaced disclosed herein may no morethan 50 degrees, 40 degrees, 30 degrees, 25 degrees, 20 degrees, 18degrees, 16 degrees, 14 degrees, 12 degrees, 10 degrees, 8 degrees, 6degrees, 4 degrees, 2 degrees, or 1 degree. In many cases the contactangle is no more than 40 degrees. Those of skill in the art will realizethat a given hydrophilic, low-binding support surface of the presentdisclosure may exhibit a water contact angle having a value of anywherewithin this range.

In some instances, the hydrophilic surfaces disclosed herein facilitatereduced wash times for bioassays, often due to reduced nonspecificbinding of biomolecules to the low-binding surfaces. In some instances,adequate wash steps may be performed in less than 60, 50, 40, 30, 20,15, 10, or less than 10 seconds. For example, in some instances adequatewash steps may be performed in less than 30 seconds.

Some low-binding surfaces of the present disclosure exhibit significantimprovement in stability or durability to prolonged exposure to solventsand elevated temperatures, or to repeated cycles of solvent exposure orchanges in temperature. For example, in some instances, the stability ofthe disclosed surfaces may be tested by fluorescently labeling afunctional group on the surface, or a tethered biomolecule (e.g., anoligonucleotide primer) on the surface, and monitoring fluorescencesignal before, during, and after prolonged exposure to solvents andelevated temperatures, or to repeated cycles of solvent exposure orchanges in temperature. In some instances, the degree of change in thefluorescence used to assess the quality of the surface may be less than1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% over a time period of 1minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 20minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 2 hours, 3hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours,15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50hours, or 100 hours of exposure to solvents and/or elevated temperatures(or any combination of these percentages as measured over these timeperiods). In some instances, the degree of change in the fluorescenceused to assess the quality of the surface may be less than 1%, 2%, 3%,4%, 5%, 10%, 15%, 20%, or 25% over 5 cycles, 10 cycles, 20 cycles, 30cycles, 40 cycles, 50 cycles, 60 cycles, 70 cycles, 80 cycles, 90cycles, 100 cycles, 200 cycles, 300 cycles, 400 cycles, 500 cycles, 600cycles, 700 cycles, 800 cycles, 900 cycles, or 1,000 cycles of repeatedexposure to solvent changes and/or changes in temperature (or anycombination of these percentages as measured over this range of cycles).

In some instances, the surfaces disclosed herein may exhibit a highratio of specific signal to nonspecific signal or other background. Forexample, when used for nucleic acid amplification, some surfaces mayexhibit an amplification signal that is at least 4, 5, 6, 7, 8, 9, 10,15, 20, 30, 40, 50, 75, 100, or greater than 100 fold greater than asignal of an adjacent unpopulated region of the surface. Similarly, somesurfaces exhibit an amplification signal that is at least 4, 5, 6, 7, 8,9, 10, 15, 20, 30, 40, 50, 75, 100, or greater than 100 fold greaterthan a signal of an adjacent amplified nucleic acid population region ofthe surface.

In some instances, fluorescence images of the disclosed low backgroundsurfaces when used in nucleic acid hybridization or amplificationapplications to create clusters of hybridized or clonally-amplifiednucleic acid molecules (e.g., that have been directly or indirectlylabeled with a fluorophore) exhibit contrast-to-noise ratios (CNRs) ofat least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 20, 210, 220, 230, 240, 250, or greater than250.

One or more types of primer may be attached or tethered to the supportsurface. In some instances, the one or more types of adapters or primersmay comprise spacer sequences, adapter sequences for hybridization toadapter-ligated target library nucleic acid sequences, forwardamplification primers, reverse amplification primers, sequencingprimers, and/or molecular barcoding sequences, or any combinationthereof. In some instances, 1 primer or adapter sequence may be tetheredto at least one layer of the surface. In some instances, at least 2, 3,4, 5, 6, 7, 8, 9, 10, or more than 10 different primer or adaptersequences may be tethered to at least one layer of the surface.

In some instances, the tethered adapter and/or primer sequences mayrange in length from about 10 nucleotides to about 100 nucleotides. Insome instances, the tethered adapter and/or primer sequences may be atleast 10, at least 20, at least 30, at least 40, at least 50, at least60, at least 70, at least 80, at least 90, or at least 100 nucleotidesin length. In some instances, the tethered adapter and/or primersequences may be at most 100, at most 90, at most 80, at most 70, atmost 60, at most 50, at most 40, at most 30, at most 20, or at most 10nucleotides in length. Any of the lower and upper values described inthis paragraph may be combined to form a range included within thepresent disclosure, for example, in some instances the length of thetethered adapter and/or primer sequences may range from about 20nucleotides to about 80 nucleotides. Those of skill in the art willrecognize that the length of the tethered adapter and/or primersequences may have any value within this range, e.g., about 24nucleotides.

In some instances, the resultant surface density of primers on the lowbinding support surfaces of the present disclosure may range from about100 primer molecules per μm² to about 100,000 primer molecules per μm².In some instances, the resultant surface density of primers on the lowbinding support surfaces of the present disclosure may range from about1,000 primer molecules per μm² to about 1,000,000 primer molecules perμm². In some instances, the surface density of primers may be at least1,000, at least 10,000, at least 100,000, or at least 1,000,000molecules per μm². In some instances, the surface density of primers maybe at most 1,000,000, at most 100,000, at most 10,000, or at most 1,000molecules per μm². Any of the lower and upper values described in thisparagraph may be combined to form a range included within the presentdisclosure, for example, in some instances the surface density ofprimers may range from about 10,000 molecules per μm² to about 100,000molecules per μm². Those of skill in the art will recognize that thesurface density of primer molecules may have any value within thisrange, e.g., about 455,000 molecules per μm². In some instances, thesurface density of target library nucleic acid sequences initiallyhybridized to adapter or primer sequences on the support surface may beless than or equal to that indicated for the surface density of tetheredprimers. In some instances, the surface density of clonally-amplifiedtarget library nucleic acid sequences hybridized to adapter or primersequences on the support surface may span the same range as thatindicated for the surface density of tethered primers.

Local densities as listed above do not preclude variation in densityacross a surface, such that a surface may comprise a region having anoligo density of, for example, 500,000/μm², while also comprising atleast a second region having a substantially different local density.

VI. Illustrative Alternative Embodiments

The disclosed methods of determining the sequence of a target nucleicacid comprise: a) contacting a double-stranded or partiallydouble-stranded target nucleic acid molecule comprising the templatestrand to be sequenced and a primer strand to be elongated with one ormore of the disclosed nucleic acid binding compositions; and b)detecting the binding of a nucleic acid binding composition to thenucleic acid molecule, thereby determining the presence of one of saidone or more nucleic acid binding compositions on said nucleic acidmolecule and the identity of the next nucleotide (i.e., the N+1 orterminal nucleotide) to be incorporated into the complementary strand.

The sequencing method may further comprise incorporating the N+1 orterminal nucleotide into the primer strand, and then repeating thecontacting, detecting, and incorporating steps for one or moreadditional iterations, thereby determining the sequence of the templatestrand of the nucleic acid molecule. After the step of detecting theternary binding complex, the primed strand of the primed target nucleicacid is extended for one base before another round of analysis isperformed. The primed target nucleic acid can be extended using theconjugated nucleotide that is attached to the polymer in the multivalentbinding composition, or using an unconjugated or untethered freenucleotide that is provided after the multivalent binding compositionhas been removed.

The extension of the primed target nucleic acid may be prevented orinhibited due to a blocked nucleotide on the strand or the use ofpolymerase that is catalytically inactive. When the nucleotide in thepolymer-nucleotide conjugate has a blocking group that prevents theextension of the nucleic acid, incorporation of a nucleotide may beachieved by the removal of a blocking group from said nucleotide (suchas by detachment of said nucleotide from its polymer, branched polymer,dendrimer, particle, or the like). When the extension of the primedtarget nucleic acid is inhibited due to the use of polymerase that iscatalytically inactive, incorporation of a nucleotide may be achieved bythe provision of a cofactor or activator such as a metal ion.

Detection of the ternary complex is achieved prior to, concurrentlywith, or following the incorporation of the nucleotide residue. In someembodiments, a primed target nucleic acid may comprise a target nucleicacid with multiple primed locations for the attachment of polymerasesand/or nucleic acid binding moieties. In some embodiments, multiplepolymerases may be attached to a single target nucleic acid molecule,such as at multiple sites within a target nucleic acid molecule. In someembodiments, multiple polymerases may be bound to a multivalent bindingcomposition disclosed herein comprising multiple nucleotides. In someembodiments, a target nucleic acid molecule may be a product of a stranddisplacement synthesis, a rolling circle amplification, a concatenationor fusion of multiple copies of a query sequence, or other such methodsas are known in the art or as are disclosed elsewhere herein to producenucleic acid molecules comprising multiple copies of an identicalsequence. Therefore, in some embodiments, multiple polymerases may beattached at multiple identical or substantially identical locationswithin a target nucleic acid which comprises multiple identical orsubstantially identical copies of a query sequence. In some embodiments,said multiple polymerases may then be involved in interactions with oneor more multivalent binding complexes; however, in preferredembodiments, the number of binding sites within a target nucleic acid isat least two, and the number of nucleotides or substrate moietiespresent on a particle-nucleotide conjugate such as a polymer-nucleotideconjugate is also greater than or equal to two.

It may be advantageous to provide the multivalent binding compositionsin combination with other elements such as to provide optimized signals,for example to provide identification of a nucleotide at a particularposition in a nucleic acid sequence. In some embodiments, thecompositions disclosed herein are provided in combination with a surfaceproviding low background binding or low levels of protein binding,especially a hydrophilic or polymer coated surface. Representativesurfaces may be found, for example, in U.S. patent application Ser. No.16/363,842, the contents of which are hereby incorporated by referencein their entirety.

In some instances, the nucleic acid molecule is tethered to the surfaceof a solid support, e.g., through hybridization of the template strandto an adapter nucleic acid sequence or primer nucleic acid sequence thatis tethered to the solid support. In some instances, the solid supportcomprises a glass, fused-silica, silicon, or polymer substrate. In someinstances, the solid support comprises a low non-specific bindingcoating comprising one or more hydrophilic polymer layers (e.g. PEGlayers) where at least one of the hydrophilic polymer layers comprises abranched polymer molecule (e.g., a branched PEG molecule comprising 4,8, 16, or 32 branches).

The solid support comprises oligonucleotide adapters or primers tetheredto at least one hydrophilic polymer layer at a surface density rangingfrom about 1,000 primer molecules per μm² to about 1,000,000 primermolecules per μm². In some instances, the surface density ofoligonucleotide primers may be at least 1,000, at least 10,000, at least100,000, or at least 1,000,000 molecules per μm². In some instances, thesurface density of oligonucleotide primers may be at most 1,000,000, atmost 100,000, at most 10,000, or at most 1,000 molecules per μm². Any ofthe lower and upper values described in this paragraph may be combinedto form a range included within the present disclosure, for example, insome instances the surface density of primers may range from about10,000 molecules per μm² to about 100,000 molecules per μm². Those ofskill in the art will recognize that the surface density of primermolecules may have any value within this range, e.g., about 455,000molecules per μm².

One of ordinary skill would recognize that in a series of iterativesequencing reactions, occasionally one or more sites will fail toincorporate a nucleotide during a given cycle, thus leading one or moresites to be unsynchronized with the bulk of the elongating nucleic acidchains. Under conditions in which sequencing signals are derived fromreactions occurring on single copies of a target nucleic acid, thesefailures to incorporate will yield discrete errors in the outputsequence. It is an object of the present disclosure to describe methodsfor reducing this type of error in sequencing reactions. For example,the use of multivalent substrates that are capable of incorporation intothe elongating strand, by providing increased probabilities of rebindingupon premature dissociation of a ternary polymerase complex, can reducethe frequency of “skipped” cycles in which a base is not incorporated.Thus, in some embodiments, the present disclosure contemplates the useof multivalent substrates as disclosed herein in which the nucleosidemoiety is comprised within a nucleotide having a free, or reversiblymodified, 5′ phosphate, diphosphate, or triphosphate moiety, and whereinthe nucleotide is connected to the particle or polymer as disclosedherein, through a labile or cleavable linkage. In some embodiments, thepresent disclosure contemplates a reduction in the intrinsic error ratedue to skipped incorporations as a result of the use of the multivalentsubstrates disclosed herein.

The present disclosure also contemplates sequencing reactions in whichsequencing signals from or relating to a given sequence are derived fromor originate within definable regions containing multiple copies of thetarget sequence. Sequencing methods incorporating multiple copies of atarget sequence have the advantage that signals can be amplified due tothe presence of multiple simultaneous sequencing reactions within thedefined region, each providing its own signal. The presence of multiplesignals within a defined area also reduces the impact of any singleskipped cycle, due to the fact that the signal from a large number ofcorrect base calls can overwhelm the signal from a smaller number ofskipped or incorrect base calls. The present disclosure furthercontemplates the inclusion of free, unlabeled nucleotides duringelongation reactions, or during a separate part of the elongation cycle,in order to provide incorporation at sites that may have been skipped inprevious cycles. For example, during or following an incorporationcycle, unlabeled blocked nucleotides may be added such that they may beincorporated at skipped sites. The unlabeled blocked nucleotides may beof the same type or types as the nucleotide attached to the multivalentbinding substrate or substrates that are or were present during aparticular cycle, or a mixture of 1, 2, 3, 4 or more types of unlabeledblocked nucleotides may be included.

When each sequencing cycle proceeds perfectly, each reaction within thedefined region will provide an identical signal. However, as notedelsewhere herein, in a series of iterative sequencing reactions,occasionally one or more sites will fail to incorporate a nucleotideduring a given cycle, thus leading one or more sites to beunsynchronized with the bulk of the elongating nucleic acid chains. Thisissue, referred to as “phasing,” leads to degradation of the sequencingsignal as the signal is contaminated with spurious signals from siteshaving skipped one or more cycles. This, in turn, creates the potentialfor errors in base identification. The progressive accumulation ofskipped cycles through multiple cycles also reduces the effective readlength, due to progressive degradation of the sequencing signal witheach cycle. It is a further object of this disclosure to provide methodsfor reducing phasing errors and/or to improve read length in sequencingreactions.

The sequencing method can include contacting a target nucleic acid ormultiple target nucleic acids, comprising multiple linked or unlinkedcopies of a target sequence, with the multivalent binding compositionsdescribed herein. Contacting said target nucleic acid, or multipletarget nucleic acids comprising multiple linked or unlinked copies of atarget sequence, with one or more particle-nucleotide conjugates mayprovide a substantially increased local concentration of the correctnucleotide being interrogated in a given sequencing cycle, thussuppressing signals from improper incorporations or phased nucleic acidchains (i.e., those elongating nucleic acid chains which have had one ormore skipped cycles).

Methods of obtaining nucleic acid sequence information can includecontacting a target nucleic acid, or multiple target nucleic acids,wherein said target nucleic acid or multiple target nucleic acidscomprise multiple linked or unlinked copies of a target sequence, withone or more particle-nucleotide conjugates. This method results in areduction in the error rate of sequencing as indicated by reduction inthe misidentification of bases, the reporting of nonexistent bases, orthe failure to report correct bases. In some embodiments, said reductionin the error orate of sequencing may comprise a reduction of 5%, 10%,15%, 20% 25%, 50%, 75%, 100%, 150%, 200%, or more compared to the errorrate observed using monovalent ligands, including free nucleotides,labeled free nucleotides, protein or peptide bound nucleotides, orlabeled protein or peptide bound nucleotides.

The method of obtaining nucleic acid sequence information can includecontacting a target nucleic acid, or multiple target nucleic acids,wherein said templet nucleic acid or multiple target nucleic acidscomprise multiple linked or unlinked copies of a target sequence, withone or more particle-nucleotide conjugates. This method results in anincrease in average read length of 5%, 10%, 15%, 20% 25%, 50%, 75%,100%, 150%, 200%, 300%, or more compared to the average read lengthobserved using monovalent ligands, including free nucleotides, labeledfree nucleotides, protein or peptide bound nucleotides, or labeledprotein or peptide bound nucleotides.

Methods of obtaining nucleic acid sequence information, said methodscomprising contacting a target nucleic acid, or multiple target nucleicacids, wherein said target nucleic acid or multiple target nucleic acidscomprise multiple linked or unlinked copies of a target sequence, withone or more particle-nucleotide conjugates. This method results in anincrease in average read length of 10NT, 20NT, 25NT, 30NT, 50NT, 75NT,100NT, 125NT, 150NT, 200NT, 250NT, 300NT, 350NT, 400NT, 500NT, or morecompared to the average read length observed using monovalent ligands,including free nucleotides, labeled free nucleotides, protein or peptidebound nucleotides, or labeled protein or peptide bound nucleotides.

The use of multivalent binding composition for sequencing effectivelyshortens the sequencing time. The sequencing reaction cycle comprisingthe contacting, detecting, and incorporating steps is performed in atotal time ranging from about 5 minutes to about 60 minutes. In someinstances, the sequencing reaction cycle is performed in at least 5minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes,at least 40 minutes, at least 50 minutes, or at least 60 minutes. Insome instances, the sequencing reaction cycle is performed in at most 60minutes, at most 50 minutes, at most 40 minutes, at most 30 minutes, atmost 20 minutes, at most 10 minutes, or at most 5 minutes. Any of thelower and upper values described in this paragraph may be combined toform a range included within the present disclosure, for example, insome instances the sequencing reaction cycle may be performed in a totaltime ranging from about 10 minutes to about 30 minutes. Those of skillin the art will recognize that the sequencing cycle time may have anyvalue within this range, e.g., about 16 minutes.

The use of multivalent binding composition for sequencing provides anmore accuracy base readout. The disclosed compositions and methods fornucleic acid sequencing will provide an average Q-score for base-callingaccuracy over a sequencing run that ranges from about 20 to about 50. Insome instances, the average Q-score is at least 20, at least 25, atleast 30, at least 35, at least 40, at least 45, or at least 50. Thoseof skill in the art will recognize that the average Q-score may have anyvalue within this range, e.g., about 32.

In some instances, the disclosed compositions and methods for nucleicacid sequencing will provide a Q-score of greater than 30 for at least50%, at least 60%, at least 70%, at least 80%, at least 85%, at least90%, at least 95%, at least 98%, or at least 99% of the terminal (orN+1) nucleotides identified. In some instances, the disclosedcompositions and methods for nucleic acid sequencing will provide aQ-score of greater than 35 for at least 50%, at least 60%, at least 70%,at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, orat least 99% of the terminal (or N+1) nucleotides identified. In someinstances, the disclosed compositions and methods for nucleic acidsequencing will provide a Q-score of greater than 40 for at least 50%,at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 98%, or at least 99% of the terminal (or N+1)nucleotides identified. In some instances, the disclosed compositionsand methods for nucleic acid sequencing will provide a Q-score ofgreater than 45 for at least 50%, at least 60%, at least 70%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least99% of the terminal (or N+1) nucleotides identified. In some instances,the disclosed compositions and methods for nucleic acid sequencing willprovide a Q-score of greater than 50 for at least 50%, at least 60%, atleast 70%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 98%, or at least 99% of the terminal (or N+1) nucleotidesidentified.

The disclosed low non-specific binding supports and associated nucleicacid hybridization and amplification methods may be used for theanalysis of nucleic acid molecules derived from any of a variety ofdifferent cell, tissue, or sample types known to those of skill in theart. For example, nucleic acids may be extracted from cells, or tissuesamples comprising one or more types of cells, derived from eukaryotes(such as animals, plants, fungi, protista), archaebacteria, oreubacteria. In some cases, nucleic acids may be extracted fromprokaryotic or eukaryotic cells, such as adherent or non-adherenteukaryotic cells. Nucleic acids are variously extracted from, forexample, primary or immortalized rodent, porcine, feline, canine,bovine, equine, primate, or human cell lines. Nucleic acids may beextracted from any of a variety of different cell, organ, or tissuetypes (e.g., white blood cells, red blood cells, platelets, epithelialcells, endothelial cells, neurons, glial cells, astrocytes, fibroblasts,skeletal muscle cells, smooth muscle cells, gametes, or cells from theheart, lungs, brain, liver, kidney, spleen, pancreas, thymus, bladder,stomach, colon, or small intestine). Nucleic acids may be extracted fromnormal or healthy cells. Alternately or in combination, acids areextracted from diseased cells, such as cancerous cells, or frompathogenic cells that are infecting a host. Some nucleic acids may beextracted from a distinct subset of cell types, e.g., immune cells (suchas T cells, cytotoxic (killer) T cells, helper T cells, alpha beta Tcells, gamma delta T cells, T cell progenitors, B cells, B-cellprogenitors, lymphoid stem cells, myeloid progenitor cells, lymphocytes,granulocytes, Natural Killer cells, plasma cells, memory cells,neutrophils, eosinophils, basophils, mast cells, monocytes, dendriticcells, and/or macrophages, or any combination thereof), undifferentiatedhuman stem cells, human stem cells that have been induced todifferentiate, rare cells (e.g., circulating tumor cells (CTCs),circulating epithelial cells, circulating endothelial cells, circulatingendometrial cells, bone marrow cells, progenitor cells, foam cells,mesenchymal cells, or trophoblasts). Other cells are contemplated andconsistent with the disclosure herein.

Nucleic acid extraction from cells or other biological samples may beperformed using any of a number of techniques known to those of skill inthe art. For example, a typical DNA extraction procedure comprises (i)collection of the cell sample or tissue sample from which DNA is to beextracted, (ii) disruption of cell membranes (i.e., cell lysis) torelease DNA and other cytoplasmic components, (iii) treatment of thelysed sample with a concentrated salt solution to precipitate proteins,lipids, and RNA, followed by centrifugation to separate out theprecipitated proteins, lipids, and RNA, and (iv) purification of DNAfrom the supernatant to remove detergents, proteins, salts, or otherreagents used during the cell membrane lysis step.

A variety of suitable commercial nucleic acid extraction andpurification kits are consistent with the disclosure herein. Examplesinclude, but are not limited to, the QIAamp kits (for isolation ofgenomic DNA from human samples) and DNAeasy kits (for isolation ofgenomic DNA from animal or plant samples) from Qiagen (Germantown, Md.),or the Maxwell® and ReliaPrep™ series of kits from Promega (Madison,Wis.).

VII. Examples 1. Preparation of Multivalent Binding Composition

One type of multi-armed substrate, as shown in FIG. 5A were made byreacting propargylamine dNTPs with Biotin-PEG-NHS. This aqueous reactionwas driven to completion and purified; resulting in a pureBiotin-PEG-dNTP species. In separate reactions, several different PEGlengths were used, varying from 1K to 20K. The Biotin-PEG-dNTP specieswere mixed with either freshly prepared or commercially sourceddye-labeled streptavidin using a Dye:SA ratio of 3-5:1. Mixing ofBiotin-PEG-dNTP with dye-labeled streptavidin was done in the presenceof excess biotin-PEG-dNTP to ensure saturation of the biotin bindingsites on each streptavidin tetramer. Complete complexes were purifiedaway from excess biotin-PEG-dNTP by size exclusion chromatography. Eachnucleotide type was conjugated and purified separately, then mixedtogether to create a 4 base mix for sequencing.

Another type of multi-armed substrate as shown in FIG. 5A was made in asingle pot by reacting multiarm PEG NHS with excess Dye-NH2 andpropargylamine dNTPs. Various multiarm PEG NHS variants were usedranging from 4-16 arms and ranging in molecular weight from 5K to 40K.After reacting, excess small molecule dye and dNTP were removed by sizeexclusion chromatography. Each nucleotide type was conjugated andpurified independently then mixed together to create a 4 base mix forsequencing.

Class II substrates as shown in FIG. 5B were made using 1 pot reactionsto simultaneously conjugate dye and dNTP. Alkyne-PEG-NHS was reactedwith excess propargylamine dNTP. This product (Alkyne-PEG-dNTP) was thenpurified to homogeneity by chromatography. Multiple PEG lengths wereused, varying between 1K and 20K. Dendrimer cores containing a variable,discrete number (12, 24, 48, 96) of azide conjugation sites. Conjugationof Alkyne-Dye and Alkyne-PEG-dNTP to the dendrimer core occurred in aone pot reaction containing excess dye and dNTP species via coppermediated click chemistry. After reacting, excess small molecule dye anddNTP were removed by size exclusion chromatography. Each nucleotide typewas conjugated and purified independently then mixed together to createa 4 base mix for sequencing. We note that this scheme allows the readysubstitution of alternative cores, such as dextrans, other polymers,proteins, etc.

Class III polymer-nucleotide conjugates as shown in FIG. 5C wereconstructed by reacting 4- or 8-arm PEG NHS with a saturating mixture ofbiotin and propargylamine dNTP. This reaction was then purified by sizeexclusion chromatography. The result of this reaction was a multiarm PEGcontaining a discrete distribution of biotin and nucleotide. Thisheterogeneous population was then reacted with dye-labeled streptavidinand purified by size exclusion chromatography. Each nucleotide type wasconjugated and purified independently then mixed together to create a 4base mix for sequencing. We note that the distribution of biotin andnucleotide is tunable by the input ration of Biotin-NH2 topropargylamine dNTP.

2. Detection of Ternary Complex

Binding reactions using the multivalent binding composition having PEGpolymer-nucleotide conjugates were analyzed to detect possible formationof ternary binding complex, and the fluorescence images of the varioussteps are illustrated in FIGS. 9A-9G. In FIG. 9A, red and greenfluorescent images post exposure of DNA rolling circle application (RCA)templates (G and A first base) to 500 nM base labeled nucleotides (A-Cy3and G-Cy5) in exposure buffer containing 20 nM Klenow polymerase and 2.5mM Sr+2. Multivalent PEG-substrate compositions were prepared usingvarying ratios of 4-armed PEG-amine (4ArmPEG-NH), biotin-PEG-amine(Biotin-PEG-NH), and nucleotide (Nuc) as follows: Samples PB1 and PB5,4ArmPEG-NH:Biotin-PEG-NH:Nuc=0.25:1:0.5; Sample PB2,4ArmPEG-NH:Biotin-PEG-NH:Nuc=0.125:0.5:0.25; Sample PB3,4ArmPEG-NH:Biotin-PEG-NH:Nuc=0.25:1:0.5. Images were collected afterwashing with imaging buffer with the same composition as the exposurebuffer, but containing no nucleotides or polymerase.

Contrast was scaled to maximize visualization of the dimmest signals,but no signals persisted following washing with imaging buffer (a.inset). In FIGS. 7B-7E, the fluorescence images showing multivalentPEG-nucleotide (base-labeled) ligands at 500 nM after mixing in theexposure buffer and imaging in the imaging buffer as above. (FIG. 9C.PB2; FIG. 9D. PB3; FIG. 9E. PB5). In FIGS. 7G-71, the fluorescenceimages showing further base discrimination by exposure of multivalentligands to inactive mutants of klenow polymerase FIG. 9G. D882H; FIG.9H. D882E; FIG. 9I. D882A, and the wild type Klenow (control) enzyme isshown in FIG. 9J.

Using multivalent ligands formulations, the base discrimination can beenabled by providing polymerase-ligand interactions having increasedavidity. In addition it is shown that increased concentration ofmultivalent ligands can generate higher signals as well as variousKlenow mutations that knock out catalytic activity can be used foravidity-based sequencing.

3. Sequencing of Target Nucleic Acid Based on Ternary Complex

In order to demonstrate sequencing based on multivalent ligandreporters, 4 known templates were amplified using RCA methods on a lowbinding substrate. Successive cycles were exposed to exposure buffercontaining 20 nM Klenow polymerase and 2.5 mM Sr⁺² and washed withimaging buffer and imaged. After imaging, the substrates were washedwith wash buffer (EDTA and high salt) and blocked nucleotides were addedto proceed to the next base. The cycle was repeated for 5 cycles. Spotswere detected using standard imaging processing and spot detection andthe sequences were called using a two color green and red scheme (G-Cy3and A-Cy5) to identify the templates being cycled. As shown in FIG. 10Aand FIG. 10B, multivalent ligands are able to provide basediscrimination through all 5 sequencing cycles.

4. Control of Nucleotide Dissociation from Ternary Complex

Ternary complexes are prepared and imaged as in Example 2. The complexesare imaged over varying lengths of time to demonstrate the persistenceof the ternary complex, e.g., as long as 60 seconds. After a length oftime, the complexes are washed with a buffer identical to the bufferused for the formation of the complexes, only lacking any divalentcation, e.g., 10 mM Tris pH 8.0, 0.5 mM EDTA, 50 mM NaCl, 0.016% TritonX100 (without SrOAc), or, alternatively, the complexes are washed with abuffer identical to the buffer used for the formation of the complexes,which contains a chelating agent but otherwise lacks any divalentcation, e.g., 10 mM Tris pH 8.0, 0.5 mM EDTA, 50 mM NaCl, 0.016% TritonX100 (without SrOAc), with 100 nm-100 mM EDTA. The fluorescence from thecomplexes is observed over time allowing observation and quantitation ofthe dissociation of the ternary complexes. A representative timecourseof this dissolution is shown in FIG. 6.

5. Extension of Target Nucleic Acid Complementary Sequence

After preparing, imaging, and dissociating ternary complexes as inExample 4, a deblocking solution is flowed into the chamber containingthe bound DNA molecules, sufficient to remove the blocking moiety, suchas an O-azidomethyl group, an O-alkyl hydroxylamino group, or an O-aminogroup, from the 3′ end of the elongating DNA strand. Either following orconcurrently with this, an extension solution is flowed into the chambercontaining the bound DNA molecules. The extension solution contains abuffer, a divalent cation sufficient to support polymerase activity, anactive polymerase, and an appropriate amount of all four nucleotides,where the nucleotides are blocked such that they are incapable ofsupporting further elongation after the addition of a single nucleotideto the elongating DNA strand, such as by incorporation of a3′-O-azidomnethyl group, a 3′-O-alkyl hydroxylamino group, or a3′-O-amino group. The elongating strand is thus extended by one and onlyone base, and the binding of catalytically inactive polymerase andmultivalent binding substrate can be used to call the next base in thecycle.

Alternatively, the nucleotides attached to the multivalent substrate maybe attached through a labile bond, such that a buffer may be flowed intothe chamber containing the bound DNA molecules containing a divalentcation or other cofactor sufficient to render the polymerasecatalytically active. Prior to, after, or concurrently with this,conditions may be provided that are sufficient to cleave the base fromthe multivalent substrate such that it may be incorporated into theelongating strand. This cleavage and incorporation causes thedissociation of the label and the polymer backbone of the multivalentsubstrate while extending the elongating DNA strand by exactly one base.Washing to remove used polymer backbone is carried out, and newmultivalent substrate is flowed into the chamber containing the boundDNA molecules, allowing the new base to be called as in Example 1.

6. Use of Polymer-Nucleotide Conjugates with Various Lengths of PEGBranch

The polymer-nucleotide conjugates having varying PEG arm lengthsdescribed in Example 3 were subjected to a single sequencing cycle andimaged as described in Example 1. As shown in FIGS. 7A-7G, increasingthe length of the PEG branches led to increased signal up to a lengthcorresponding to an apparent average PEG MW of 5K (FIGS. 7A-7D). The useof longer PEG arms than this led to decreases in the fluorescence signalfor both Cy3-A and Cy5-G (FIG. 7E-7G). Quantitative measurements ofsignal intensity are shown graphically in FIG. 8.

7. Enhancement of Multivalent Substrate Binding by Addition of Detergent

Multivalent substrates were prepared and assembled into bindingcomplexes in the presence and absence of detergent: one set using 10 mMTris pH 8.0, 0.5 mM EDTA, 50 mM NaCl, 5 mM SroAc, 0% TritonX100(Condition A), and one set using 10 mM Tris pH 8.0, 0.5 mM EDTA, 50 mMNaCl, 5 mM SroAc, 0.016% Triton X100. FIG. 11 shows normalizedfluorescence from these multivalent substrates bound to DNA clusters,with the substrate complexes formed in the presence (condition B) ofTriton-X100 (0.016%) showing clearly enhanced fluorescence intensity.

8. Evaluation of Multivalent Substrate Binding Timecourses

Multivalent substrates were prepared and assembled into bindingcomplexes as in Example 2. Complexes were also formed under identicalbuffer conditions using free labeled nucleotides. Complexes were imagedover the course of 60 min. to characterize the persistence time of thecomplexes. FIG. 12 shows representative results. Multivalent bindingcomplexes are stable over timescales of >60 minutes (FIG. 12, bottom)while labeled free nucleotides dissociate in less than one minute (FIG.12, top).

VIII. Conclusion

The present inventions provide greatly improved methods and compositionsfor DNA sequencing and biosensor applications. It is to be understoodthat the above description is intended to be illustrative and notrestrictive. Many embodiments will be apparent to those of skill in theart upon reviewing the above description. By way of example, theinvention has been described primarily with reference to the use ofpolymer-nucleotide conjugate, but it will be readily recognized by thoseof skill in the art that other types of particle-nucleotide conjugatescould also be used. For example, in some embodiments it may be desirableto use particle-nucleotide conjugates which include quantum dot; aliposome; or an emulsion particle. Alternatively, the conjugation couldbe achieved by noncovalent bond such as hydrogen bond or otherinteractions. The scope of the invention should, therefore, bedetermined not with reference to the above description, but shouldinstead be determined with reference to the appended claims, along withthe full scope of equivalents to which such claims are entitled.

1. A method of determining an identity of a nucleotide in a nucleic acidsequence, the method comprising: (a) providing a primed nucleic acidsequence comprising a nucleobase coupled to a nucleotide of said primednucleic acid sequence at an N position, wherein said nucleobasecomprises a blocking group coupled thereto; (b) bringing said primednucleic acid sequence into contact with a conjugated polymer-nucleotidecomposition under conditions sufficient to form a stable ternary complexbetween a nucleotide moiety of said conjugated polymer-nucleotidecomposition and said primed nucleic acid sequence at an N+1 position;and (c) detecting said stable ternary complex to determine said identityof said nucleotide at said N+1 position of said primed nucleic acidsequence.
 2. The method of claim 1, wherein said conjugatedpolymer-nucleotide composition comprises a plurality of said nucleotidemoiety conjugated to a polymer core.
 3. The method of claim 2, whereinsaid polymer core comprises polyethylene glycol, polypropylene glycol,polyvinyl acetate, polylactic acid, or polyglycolic acid.
 4. The methodof claim 3, wherein said polymer core is a branched.
 5. The method ofclaim 1, wherein said conjugated polymer-nucleotide further comprises ainteraction moiety coupled to said polymer core, wherein saidinteraction moiety is selected from the group consisting of avidin,streptavidin, a biotin moiety, an affinity tag, an antibody orantigen-binding fragment thereof, and a receptor.
 6. The method of claim1, wherein said conjugated polymer-nucleotide composition comprises adetectable label.
 7. The method of claim 6, wherein said detectablelabel comprises a fluorescent label, a fluorescence resonance energytransfer (FRET) donor, or a fluorescence resonance energy transfer(FRET) acceptor.
 8. The method of claim 1, wherein said blocking groupis coupled to said nucleobase at the 3′ carbon.
 9. The method of claim1, wherein said blocking group is selected from the group consisting ofO-alkyl hydroxylamine, O-methyl, 3′-phosphorothioate, a 3′-O-malonyl,and a 3′-O-benzyl.
 10. The method of claim 1, wherein said conjugatedpolymer-nucleotide composition comprises a plurality of detectablelabels.
 11. The method of claim 10, wherein said plurality of detectablelabels comprises a fluorescent label, a fluorescence resonance energytransfer (FRET) donor, or a fluorescence resonance energy transfer(FRET) acceptor.
 12. The method of claim 1, further comprising, before(a): (d) amplifying a plurality of nucleic acid sequences; and (e)contacting said plurality of nucleic acid sequences with a primer toform a library of said primed nucleic acid sequence.
 13. The method ofclaim 12, wherein said amplifying is performed using polymerase chainreaction (PCR), multiple displacement amplification (MDA),transcription-mediated amplification (TMA), nucleic acid sequence-basedamplification (NASBA), strand displacement amplification (SDA),real-time SDA, bridge amplification, isothermal bridge amplification,rolling circle amplification (RCA), circle-to-circle amplification,helicase-dependent amplification, recombinase-dependent amplification,single-stranded binding (SSB) protein-dependent amplification, or anycombination thereof.
 14. The method of claim 13, wherein said amplifyingcomprises RCA.
 15. The method of claim 1, further comprising, following(b) and (c): (d) deblocking said nucleobase comprising said blockinggroup coupled thereto at said N position; and (e) extending said primednucleic acid sequence by incorporating a second nucleobase comprising asecond blocking group coupled thereto at said N+1 position.
 16. Themethod of claim 15, wherein said deblocking comprises bringing saidprimed nucleic acid sequence into contact with a deblocking solutionunder conditions sufficient to remove said blocking group from saidnucleobase.
 17. The method of claim 15, wherein said extending comprisesbringing said primed nucleic acid sequence into contact with anextension solution under conditions sufficient to incorporate saidsecond nucleobase into said primed nucleic acid sequence at said N+1position.
 18. The method of claim 17, wherein said extension solutioncomprises: (i) a polymerizing enzyme that is catalytically active; and(ii) a plurality of said second nucleobase comprising said secondblocking group coupled thereto.
 19. The method of claim 18, wherein saidplurality of said second nucleobase comprises a plurality types ofnucleobases selected from the group consisting of adenine, cytosine,thymine, and guanine.
 20. The method of claim 15, wherein said secondblocking group is selected from the group consisting of O-alkylhydroxylamine, O-methyl, 3′-phosphorothioate, a 3′-O-malonyl, and a3′-O-benzyl.